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Sample records for marrow stromal osteoblast

  1. Effects of biodegradable polymer particles on rat marrow-derived stromal osteoblasts in vitro.

    PubMed

    Wake, M C; Gerecht, P D; Lu, L; Mikos, A G

    1998-07-01

    Effects of biodegradable particles of poly(L-lactic acid) (PLLA) and poly(DL-lactic-co-glycolic acid) (PLGA) 50/50 with diameter ranging from 1.0 to 1.5 microm on rat marrow stromal osteoblasts in vitro have been investigated over a period of 28 days. This study examined the effects of three particle parameters, concentration, polymer molecular weight, and composition, on osteoblast proliferation and function. Cell cultures were challenged with particles at two different time points: upon cell seeding (Day 1), and after cells had begun to establish their own mineralized extracellular matrix (Day 14). The most significant trend observed in those cultures challenged with particles beginning on Day 1 was due to increasing the concentration of particles, resulting in decreased [3H]-thymidine incorporation, cell count, and mineralization. Those cultures challenged with particles beginning on Day 14 were significantly more mineralized than those challenged with particles beginning on Day 1. In addition, increasing osteocalcin secretion confirmed the osteoblastic phenotype of the derived stromal cells. These studies suggest that the particles may affect the bone remodeling process surrounding a degrading implant by direct interaction with osteoblasts in addition to their indirect contributions to the inflammatory mechanism via mediators secreted by macrophages upon their phagocytosis.

  2. TGF-beta1 release from biodegradable polymer microparticles: its effects on marrow stromal osteoblast function

    NASA Technical Reports Server (NTRS)

    Lu, L.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    BACKGROUND: Controlled release of transforming growth factor-beta1 (TGF-beta1) to a bone defect may be beneficial for the induction of a bone regeneration cascade. The objectives of this work were to assess the feasibility of using biodegradable polymer microparticles as carriers for controlled TGF-beta1 delivery and the effects of released TGF-beta1 on the proliferation and differentiation of marrow stromal cells in vitro. METHODS: Recombinant human TGF-beta1 was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG). Fluorescein isothiocynate-labeled bovine serum albumin (FITC-BSA) was co-encapsulated as a porogen. The effects of PEG content (0, 1, or 5% by weight [wt%]) and buffer pH (3, 5, or 7.4) on the protein release kinetics and the degradation of PLGA were determined in vitro for as long as 28 days. Rat marrow stromal cells were seeded on a biodegradable poly(propylene fumarate) (PPF) substrate. The dose response and biological activity of released TGF-beta1 was determined after 3 days in culture. The effects of TGF-beta1 released from PLGA/PEG microparticles on marrow stromal cell proliferation and osteoblastic differentiation were assessed during a 21-day period. RESULTS: TGF-beta1 was encapsulated along with FITC-BSA into PLGA/PEG blend microparticles and released in a multiphasic fashion including an initial burst for as long as 28 days in vitro. Increasing the initial PEG content resulted in a decreased cumulative mass of released proteins. Aggregation of FITC-BSA occurred at lower buffer pH, which led to decreased release rates of both proteins. The degradation of PLGA was increased at higher PEG content and significantly accelerated at acidic pH conditions. Rat marrow stromal cells cultured on PPF substrates showed a dose response to TGF-beta1 released from the microparticles similar to that of added TGF-beta1, indicating that the activity of TGF-beta1 was retained during microparticle

  3. Osteochondral interface generation by rabbit bone marrow stromal cells and osteoblasts coculture.

    PubMed

    Chen, Kelei; Teh, Thomas Kok Hiong; Ravi, Sujata; Toh, Siew Lok; Goh, James Cho Hong

    2012-09-01

    Physiological osteochondral interface regeneration is a significant challenge. This study aims to investigate the effect of the coculture of chondrogenic rabbit bone marrow stromal cells (rBMSCs) with rabbit osteoblasts in a specially designed two-dimensional (2D)-three-dimensional (3D) co-interface culture to develop the intermediate osteochondral region in vitro. The 2D-3D coculture system was set up by first independently culturing chondrogenic rBMSCs on a scaffold and osteoblasts in cell culture plates, and subsequently placed in contact and cocultured. As control, samples not cocultured with osteoblasts were used. The regulatory effects exerted by osteoblasts on chondrogenic rBMSCs were quantified by real-time polymerase chain reaction. To study the effect of coculture on cells located in different parts of the scaffold, samples were separated into two parts and significantly different gene expression patterns were found between them. In comparison with the control group, a significant moderate downregulation of chondrogenic marker genes, such as Collagen II and Aggrecan was observed. However, the Sox-9 and Collagen I expression increased. More importantly, chondrogenic rBMSCs in the coculture system were shown to form the osteochondral interface layer by expressing calcified cartilage zone specific extracellular matrix marker Collagen X and the hypertrophic chondrocyte marker MMP-13, which were not observed in the control group. Specifically, only the chondrogenic rBMSC layer in contact with the osteoblasts expressed Collagen X and MMP-13, indicating the positive influence of the coculture upon interface formation. Biochemical analyses, histology results, and immunohistochemical staining further supported this observation. In conclusion, this study revealed that specific regulatory stimulations from osteoblasts in the 2D-3D interface coculture system could induce the formation of ostochondral interface for the purpose of osteochondral tissue engineering.

  4. Melphalan exposure induces an Interleukin-6 deficit in bone marrow stromal cells and osteoblasts

    PubMed Central

    Rellick, Stephanie L.; Piktel, Debbie; Walton, Cheryl; Hall, Brett; Petros, William; Fortney, James E.; Gencheva, Marieta; Denvir, Jim; Hobbs, Gerald; Craig, Michael; Gibson, Laura F.

    2012-01-01

    Bone marrow stromal cells (BMSC) and osteoblasts are critical components of the microenvironment that support hematopoietic recovery following bone marrow transplantation. Aggressive chemotherapy not only affects tumor cells, but also influences additional structural and functional components of the microenvironment. Successful reconstitution of hematopoiesis following stem cell or bone marrow transplantation after aggressive chemotherapy is dependent upon components of the microenvironment maintaining their supportive function. This includes secretion of soluble factors and expression of cellular adhesion molecules that impact on development of hematopoietic cells. In the current study, we investigated the effects of chemotherapy treatment on BMSC and human osteoblast (HOB) expression of Interleukin-6 (IL-6) as one regulatory factor. IL-6 is a pleiotrophic cytokine which has diverse effects on hematopoietic cell development. In the current study we demonstrate that exposure of BMSC or HOB to melphalan leads to decreases in IL-6 protein expression. Decreased IL-6 protein is the most pronounced following melphalan exposure compared to several other chemotherapeutic agents tested. We also observed that melphalan decreased IL-6 mRNA in both BMSC and HOB. Finally, using a model of BMSC or HOB co-cultured with myeloma cells exposed to melphalan, we observed that IL-6 protein was also decreased, consistent with treatment of adherent cells alone. Collectively, these observations are of dual significance. First, suggesting that chemotherapy induced IL-6 deficits in the bone marrow occur which may result in defective hematopoietic support of early progenitor cells. In contrast, the decrease in IL-6 protein may be a beneficial mechanism by which melphalan acts as a valuable therapeutic agent for treatment of multiple myeloma, where IL-6 present in the bone marrow acts as a proliferative factor and contributes to disease progression. Taken together, these data emphasize the

  5. Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures.

    PubMed

    Leboy, P S; Beresford, J N; Devlin, C; Owen, M E

    1991-03-01

    We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the

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

    PubMed Central

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

    2016-01-01

    The molecular mechanisms controlling the differentiation of bone marrow stromal stem cells into osteoblasts remain largely unknown. In this study, we investigated whether bone marrow stromal antigen 2 (BST2) influences differentiation toward the osteoblasts lineage. BST2 mRNA expression in human alveolar-derived bone marrow stromal cells (hAD-BMSCs) increased during differentiation into osteoblasts. hAD-BMSCs differentiation into osteoblasts and the mRNA expression of the bone-specific markers alkaline phosphatase, collagen type α 1, bone sialoprotein, osteocalcin, and osterix were reduced by BST2 knockdown using siRNA. Furthermore, BST2 knockdown in hAD-BMSCs resulted in decreased RUNX2 mRNA and protein expression. We hypothesized that BST2 is involved in differentiation of into osteoblasts via the BMP2 signaling pathway. Accordingly, we evaluated the mRNA expression levels of BMP2, BMP receptors (BMPR1 and 2), and the downstream signaling molecules SMAD1, SMAD4, and p-SMAD1/5/8 in BST2 knockdown cells. BMP2 expression following the induction of differentiation was significantly lower in BST2 knockdown cells than in cells treated with a non-targeting control siRNA. Similar results were found for the knockdown of the BMP2 receptor- BMPR1A. We also identified significantly lower expression of SMAD1, SMAD4, and p-SMAD1/5/8 in the BST2 knockdown cells than control cells. Our data provide the first evidence that BST2 is involved in the osteogenic differentiation of bone marrow stromal cells via the regulation of the BMP2 signaling pathway. PMID:27359105

  7. The effect of different implant biomaterials on the behavior of canine bone marrow stromal cells during their differentiation into osteoblasts.

    PubMed

    Özdal-Kurt, F; Tuğlu, I; Vatansever, H S; Tong, S; Şen, B H; Deliloğlu-Gürhan, S I

    2016-08-01

    We investigated the effects of different implant biomaterials on cultured canine bone marrow stromal cells (BMSC) undergoing differentiation into osteoblasts (dBMSC). BMSC were isolated from canine humerus by marrow aspiration, cultured and differentiated on calcium phosphate scaffold (CPS), hydroxyapatite, hydroxyapatite in gel form and titanium mesh. We used the MTT method to determine the effects of osteogenic media on proliferation. The characteristics of dBMSC were assessed using alizarin red (AR), immunocytochemistry and osteoblastic markers including alkaline phosphatase/von Kossa (ALP/VK), osteocalcin (OC) and osteonectin (ON), and ELISA. The morphology of dBMSC on the biomaterials was investigated using inverted phase contrast microscopy and scanning electron microscopy. We detected expression of ALP/VK, AR, OC and ON by day 7 of culture; expression increased from day 14 until day 21. CPS supported the best adhesion, cell spreading, proliferation and differentiation of BMSCs. The effects of the biomaterials depended on their surface properties. Expression of osteoblastic markers showed that canine dBMSCs became functional osteoblasts. Tissue engineered stem cells can be useful clinically for autologous implants for treating bone wounds.

  8. Osteoblasts and Bone Marrow Mesenchymal Stromal Cells Control Hematopoietic Stem Cell Migration and Proliferation in 3D In Vitro Model

    PubMed Central

    de Barros, Ana Paula D. N.; Takiya, Christina M.; Garzoni, Luciana R.; Leal-Ferreira, Mona Lisa; Dutra, Hélio S.; Chiarini, Luciana B.; Meirelles, Maria Nazareth; Borojevic, Radovan; Rossi, Maria Isabel D.

    2010-01-01

    Background Migration, proliferation, and differentiation of hematopoietic stem cells (HSCs) are dependent upon a complex three-dimensional (3D) bone marrow microenvironment. Although osteoblasts control the HSC pool, the subendosteal niche is complex and its cellular composition and the role of each cell population in HSC fate have not been established. In vivo models are complex and involve subtle species-specific differences, while bidimensional cultures do not reflect the 3D tissue organization. The aim of this study was to investigate in vitro the role of human bone marrow–derived mesenchymal stromal cells (BMSC) and active osteoblasts in control of migration, lodgment, and proliferation of HSCs. Methodology/Principal Findings A complex mixed multicellular spheroid in vitro model was developed with human BMSC, undifferentiated or induced for one week into osteoblasts. A clear limit between the two stromal cells was established, and deposition of extracellular matrix proteins fibronectin, collagens I and IV, laminin, and osteopontin was similar to the observed in vivo. Noninduced BMSC cultured as spheroid expressed higher levels of mRNA for the chemokine CXCL12, and the growth factors Wnt5a and Kit ligand. Cord blood and bone marrow CD34+ cells moved in and out the spheroids, and some lodged at the interface of the two stromal cells. Myeloid colony-forming cells were maintained after seven days of coculture with mixed spheroids, and the frequency of cycling CD34+ cells was decreased. Conclusions/Significance Undifferentiated and one-week osteo-induced BMSC self-assembled in a 3D spheroid and formed a microenvironment that is informative for hematopoietic progenitor cells, allowing their lodgment and controlling their proliferation. PMID:20161704

  9. A cost-effective method to enhance adenoviral transduction of primary murine osteoblasts and bone marrow stromal cells

    PubMed Central

    Buo, Atum M; Williams, Mark S; Kerr, Jaclyn P; Stains, Joseph P

    2016-01-01

    We report here a method for the use of poly-l-lysine (PLL) to markedly improve the adenoviral transduction efficiency of primary murine osteoblasts and bone marrow stromal cells (BMSCs) in culture and in situ, which are typically difficult to transduce. We show by fluorescence microscopy and flow cytometry that the addition of PLL to the viral-containing medium significantly increases the number of green fluorescence protein (GFP)-positive osteoblasts and BMSCs transduced with an enhanced GFP-expressing adenovirus. We also demonstrate that PLL can greatly enhance the adenoviral transduction of osteoblasts and osteocytes in situ in ex vivo tibia and calvaria, as well as in long bone fragments. In addition, we validate that PLL can improve routine adenoviral transduction studies by permitting the use of low multiplicities of infection to obtain the desired biologic effect. Ultimately, the use of PLL to facilitate adenoviral gene transfer in osteogenic cells can provide a cost-effective means of performing efficient gene transfer studies in the context of bone research. PMID:27547486

  10. Marrow stromal osteoblast function on a poly(propylene fumarate)/beta-tricalcium phosphate biodegradable orthopaedic composite.

    PubMed

    Peter, S J; Lu, L; Kim, D J; Mikos, A G

    2000-06-01

    The objective of this study was to assess the osteoconductivity of a poly(propylene fumarate)/beta-tricalcium phosphate (PPF/beta-TCP) composite in vitro. We examined whether primary rat marrow stromal cells would attach, proliferate, and express differentiated osteoblastic function when seeded on PPF/beta-TCP substrates. Attachment studies showed that a confluent monolayer of cells had adhered to the substrates within an 8 h time frame for marrow stromal cells seeded at confluent numbers. Proliferation and differentiated function of the cells were then investigated for a period of 4 weeks for an initial seeding density of 42,000 cells/cm2. Rapid proliferation during the first 24 h as determined by 3H-thymidine incorporation was mirrored by an initial rapid increase in total cell number by DNA assay. A lower proliferation rate and a gradual increase in cell number persisted for the remainder of the study, resulting in a final cell number of 128,000 cells/cm2. Differentiated cell function was assessed by measuring alkaline phosphatase (ALP) activity and osteocalcin (OC) production throughout the time course. Both markers of osteoblastic differentiation increased significantly over a 4-week period. By day 28, cells grown on PPF/beta-TCP reached a maximal ALP activity of 11 (+/- 1) x 10(-7) micromol/min/cell, while the OC production reached 40 (+/- 1) x 10(-6) ng/cell. These data show that a PPF/beta-TCP composite exhibits in vitro osteoconductivity similar to or better than that of control tissue culture polystyrene.

  11. Craniosynostosis-Associated Fgfr2C342Y Mutant Bone Marrow Stromal Cells Exhibit Cell Autonomous Abnormalities in Osteoblast Differentiation and Bone Formation

    PubMed Central

    Liu, J.; Kwon, T.-G.; Nam, H. K.; Hatch, N. E.

    2013-01-01

    We recently reported that cranial bones of Fgfr2C342Y/+ craniosynostotic mice are diminished in density when compared to those of wild type mice, and that cranial bone cells isolated from the mutant mice exhibit inhibited late stage osteoblast differentiation. To provide further support for the idea that craniosynostosis-associated Fgfr mutations lead to cell autonomous defects in osteoblast differentiation and mineralized tissue formation, here we tested bone marrow stromal cells isolated from Fgfr2C342Y/+ mice for their ability to differentiate into osteoblasts. Additionally, to determine if the low bone mass phenotype of Crouzon syndrome includes the appendicular skeleton, long bones were assessed by micro CT. Fgfr2C342Y/+ cells showed increased osteoblastic gene expression during early osteoblastic differentiation but decreased expression of alkaline phosphatase mRNA and enzyme activity, and decreased mineralization during later stages of differentiation, when cultured under 2D in vitro conditions. Cells isolated from Fgfr2C342Y/+ mice also formed less bone when allowed to differentiate in a 3D matrix in vivo. Cortical bone parameters were diminished in long bones of Fgfr2C342Y/+ mice. These results demonstrate that marrow stromal cells of Fgfr2C342Y/+ mice have an autonomous defect in osteoblast differentiation and bone mineralization, and that the Fgfr2C342Y mutation influences both the axial and appendicular skeletons. PMID:23762837

  12. Role of P2 × 7 receptor in the differentiation of bone marrow stromal cells into osteoblasts and adipocytes.

    PubMed

    Li, Wenkai; Li, Guizhen; Zhang, Yingchi; Wei, Sheng; Song, Mingyu; Wang, Wei; Yuan, Xuefeng; Wu, Hua; Yang, Yong

    2015-12-10

    Imbalance in osteogenesis and adipogenesis of bone marrow stromal cells is a crucial pathological process of osteoporosis. P2 × 7-deficient mice were previously shown to exhibit an osteopenic phenotype and abnormal fat distribution, leading us to hypothesize that P2 × 7R activation was involved in the differentiation of BMSCs. Consequently, we investigated the effect of P2 × 7R activation on osteogenic and adipogenic differentiation of BMSCs in vitro, and established an ovariectomized (OVX) osteoporosis model to test P2 × 7R activation on adipocytes formation, trabecular and cortical bone parameters in vivo. Our results showed that activation of P2 × 7R by BzATP resulted in increase in the gene expression of osteoblastic markers, the activity of alkaline phosphatase and bone mineralization, and decrease in the gene expression of adipogenic markers and the number of adipocytes generated by BMSCs. MicroCT analysis showed that BzATP treatment ameliorated the micro-architecture of trabecular bones in OVX mice, while cortical bone parameters were unaffected. H&E staining analysis showed that was increase in the volume of trabecular bone and number of trabecular bone, and decrease in bone marrow adipocytes in BzATP-treated OVX mice compared with OVX mice. Also, activation of P2 × 7R transduced to ERK1/2 and JNK signaling pathways. This transduction was prevented by BBG, U0126, and SP600125. U0126 and SP600125 prevented BzATP-induced up-regulation of osteogenic-related genes expression and down-regulation of adipogenic-related genes expression. These data suggest that BzATP activates the differentiation of BMSCs into osteoblasts but not adipocytes by stimulating ERK1/2 and JNK signaling pathways in a P2 × 7R-dependent way. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Bone marrow stromal/stem cell-derived extracellular vesicles regulate osteoblast activity and differentiation in vitro and promote bone regeneration in vivo.

    PubMed

    Qin, Yunhao; Wang, Lian; Gao, Zhengliang; Chen, Genyin; Zhang, Changqing

    2016-02-25

    Emerging evidence suggests that extracellular vesicles (EVs) are secreted by diverse tissues and play important roles in cell-cell communication, organ interactions and tissue homeostasis. Studies have reported the use of EVs to stimulate tissue regeneration, such as hepatic cell regeneration, and to treat diseases, such as pulmonary hypertension. However, little is known about the osteogenic effect of EVs. In this study, we explore the role of bone marrow stromal cell-derived EVs in the regulation of osteoblast activity and bone regeneration. We isolated bone marrow stromal/stem cell (BMSC)-derived EVs through gradient ultracentrifugation and ultrafiltration, and tested the influence of the EVs on osteogenesis both in vivo and in vitro. The results indicated that EVs positively regulated osteogenic genes and osteoblastic differentiation but did not inhibit proliferation in vitro. Furthermore, we constructed an EVs delivery system to stimulate bone formation in Sprague Dawley (SD) rats with calvarial defects. We found that BMSC-derived EVs led to more bone formation in the critical-size calvarial bone defects. Moreover, we found that miR-196a plays an essential role in the regulation of osteoblastic differentiation and the expression of osteogenic genes. We anticipate that our assay using bone marrow stromal cell-derived EVs will become a valuable tool for promoting bone regeneration.

  14. Up-regulation of BMP2/4 signaling increases both osteoblast-specific marker expression and bone marrow adipogenesis in Gja1Jrt/+ stromal cell cultures.

    PubMed

    Zappitelli, Tanya; Chen, Frieda; Aubin, Jane E

    2015-03-01

    Gja1(Jrt)/+ mice carry a mutation in one allele of the gap junction protein α1 gene (Gja1), resulting in a G60S connexin 43 (Cx43) mutant protein that is dominant negative for Cx43 protein production of <50% of wild-type (WT) levels and significantly reduced gap junction formation and function in osteoblasts and other Cx43-expressing cells. Previously we reported that Gja1(Jrt)/+ mice exhibited early-onset osteopenia caused by activation of osteoclasts secondary to activation of osteoblast lineage cells, which expressed increased RANKL and produced an abnormal resorption-stimulating bone matrix high in BSP content. Gja1(Jrt)/+ mice also displayed early and progressive bone marrow atrophy, with a significant increase in bone marrow adiposity versus WT littermates but no increase in adipose tissues elsewhere in the body. BMP2/4 production and signaling were increased in Gja1(Jrt)/+ trabecular bone and osteogenic stromal cell cultures, which contributed to the up-regulated expression of osteoblast-specific markers (e.g., Bsp and Ocn) in Gja1(Jrt)/+ osteoblasts and increased Pparg2 expression in bone marrow-derived adipoprogenitors in vitro. The elevated levels of BMP2/4 signaling in G60S Cx43-containing cells resulted at least in part from elevated levels of cAMP. We conclude that up-regulation of BMP2/4 signaling in trabecular bone and/or stromal cells increases osteoblast-specific marker expression in hyperactive Gja1(Jrt)/+ osteoblasts and may also increase bone marrow adipogenesis by up-regulation of Pparg2 in the Cx43-deficient Gja1(Jrt)/+ mouse model.

  15. Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 1. Encapsulation of marrow stromal osteoblasts in surface crosslinked gelatin microparticles.

    PubMed

    Payne, Richard G; Yaszemski, Michael J; Yasko, Alan W; Mikos, Antonios G

    2002-11-01

    This study investigated the temporary encapsulation of rat marrow stromal osteoblasts in surface crosslinked gelatin microparticles. Cells were encapsulated in uncrosslinked gelatin microparticles of average diameter of 630 microm containing approximately 53 cells. Gelatin microparticles were crosslinked to shell thicknesses of approximately 75 microm via exposure to 1 mM dithiobis(succinimidylpropionate) (DSP) solution for 15 min or 5 mm DSP solution for 5 min for the production of microparticles dispersing approximately 60 min after placement into a physiologic fluid at 37 degrees C. Formed microparticles were placed into culture wells at a cell seeding density of 5.3 x 10(4) cells/cm2 and, following the degradation and/or dissolution of gelatin, the cells were cultured in the presence of osteogenic supplements for 28 days. Samples were taken at specified time points and analyzed by a DNA assay for cell number and a 3H-thymidine incorporation assay for proliferative potential. Samples were also obtained and analyzed at several time points by alkaline phosphatase, osteocalcin, and mineralization assays for early and late phenotypic expression markers of osteoblastic differentiation. The measurements from the different assays for encapsulated cells (EC) in uncrosslinked and crosslinked gelatin microparticles were normalized with the cell numbers from the DNA assay and compared with those for nonencapsulated control cells. The results demonstrated that the marrow stromal cells survived the encapsulation procedure in uncrosslinked gelatin microparticles and also retained their proliferative potential and osteoblastic phenotype over a 28 day period, although at a slightly lower level than the nonencapsulated cells. The results further showed that the marrow stromal cells survived the encapsulation in crosslinked gelatin microparticles prepared via exposure to 5mm DSP for 5 min and also retained their proliferative potential and osteoblastic phenotype over a 28 day

  16. Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

    NASA Technical Reports Server (NTRS)

    Bancroft, Gregory N.; Sikavitsas, Vassilios I.; van den Dolder, Juliette; Sheffield, Tiffany L.; Ambrose, Catherine G.; Jansen, John A.; Mikos, Antonios G.; McIntire, L. V. (Principal Investigator)

    2002-01-01

    Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

  17. Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

    PubMed Central

    Bancroft, Gregory N.; Sikavitsas, Vassilios I.; van den Dolder, Juliette; Sheffield, Tiffany L.; Ambrose, Catherine G.; Jansen, John A.; Mikos, Antonios G.

    2002-01-01

    Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering. PMID:12242339

  18. Osteoblastic phenotype of rat marrow stromal cells cultured in the presence of dexamethasone, beta-glycerolphosphate, and L-ascorbic acid

    NASA Technical Reports Server (NTRS)

    Peter, S. J.; Liang, C. R.; Kim, D. J.; Widmer, M. S.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    We investigated the effects of the time course of addition of osteogenic supplements dexamethasone, beta-glycerolphosphate, and L-ascorbic acid to rat marrow stromal cells, and the exposure time on the proliferation and differentiation of the cells. It was the goal of these experiments to determine the time point for supplement addition to optimize marrow stromal cell proliferation and osteoblastic differentiation. To determine this, two studies were performed; one study was based on the age of the cells from harvest, and the other study was based on the duration of exposure to supplemented medium. Cells were seen to proliferate rapidly at early time points in the presence and absence of osteogenic supplements as determined by 3H-thymidine incorporation into the DNA of replicating cells. These results were supported by cell counts ascertained through total DNA analysis. Alkaline phosphatase (ALP) activity and osteocalcin production at 21 days were highest for both experimental designs when the cells were exposed to supplemented medium immediately upon harvest. The ALP levels at 21 days were six times greater for cells maintained in supplements throughout than for control cells cultured in the absence of supplements for both studies, reaching an absolute value of 75 x 10(-7) micromole/min/cell. Osteocalcin production reached 20 x 10(-6) ng/cell at 21 days in both studies for cells maintained in supplemented medium throughout the study, whereas the control cells produced an insignificant amount of osteocalcin. These results suggest that the addition of osteogenic supplements to marrow-derived cells early in the culture period did not inhibit proliferation and greatly enhanced the osteoblastic phenotype of cells in a rat model.

  19. Osteoblastic phenotype of rat marrow stromal cells cultured in the presence of dexamethasone, beta-glycerolphosphate, and L-ascorbic acid

    NASA Technical Reports Server (NTRS)

    Peter, S. J.; Liang, C. R.; Kim, D. J.; Widmer, M. S.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    We investigated the effects of the time course of addition of osteogenic supplements dexamethasone, beta-glycerolphosphate, and L-ascorbic acid to rat marrow stromal cells, and the exposure time on the proliferation and differentiation of the cells. It was the goal of these experiments to determine the time point for supplement addition to optimize marrow stromal cell proliferation and osteoblastic differentiation. To determine this, two studies were performed; one study was based on the age of the cells from harvest, and the other study was based on the duration of exposure to supplemented medium. Cells were seen to proliferate rapidly at early time points in the presence and absence of osteogenic supplements as determined by 3H-thymidine incorporation into the DNA of replicating cells. These results were supported by cell counts ascertained through total DNA analysis. Alkaline phosphatase (ALP) activity and osteocalcin production at 21 days were highest for both experimental designs when the cells were exposed to supplemented medium immediately upon harvest. The ALP levels at 21 days were six times greater for cells maintained in supplements throughout than for control cells cultured in the absence of supplements for both studies, reaching an absolute value of 75 x 10(-7) micromole/min/cell. Osteocalcin production reached 20 x 10(-6) ng/cell at 21 days in both studies for cells maintained in supplemented medium throughout the study, whereas the control cells produced an insignificant amount of osteocalcin. These results suggest that the addition of osteogenic supplements to marrow-derived cells early in the culture period did not inhibit proliferation and greatly enhanced the osteoblastic phenotype of cells in a rat model.

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

    PubMed Central

    Rifkin, Daniel; Dabovic, Branka

    2017-01-01

    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. PMID:28512472

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

    PubMed

    Selvamurugan, Nagarajan; He, Zhiming; Rifkin, Daniel; Dabovic, Branka; Partridge, Nicola C

    2017-01-01

    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.

  2. The effects of retinoic acid on reversing the adipocyte differentiation into an osteoblastic tendency in ST2 cells, a murine bone marrow-derived stromal cell line.

    PubMed

    Ding, J; Woo, J T; Nagai, K

    2001-07-01

    Although the mouse bone marrow stromal cell line ST2 has been known to be differentiated into osteoblasts, the differentiation characteristics of the cell into adipocyte and the concerned relationship between its adipogenesis and osteogenesis remains unknown. The adipogenic induction medium which is made up of insulin, dexamethasone (DEX) and 3-isobutyl-1-methylxanthine(IBMX), stimulated the expression of n early adipogenic marker PPAR gamma and a late marker GPDH in ST2 cells. The triglyceride accumulation and lipid stain level generated by the induction medium in ST2 cells was inhibited by RA with IC(50) at about 1 nM. The induction medium up-regulated expression of PPARgamma and GPDH was also inhibited by RA whereas RA (30 nM) exterted no effect on the cell growth. Interestingly, treatment of the cells with induction medium in the presense of RA caused a 3- or 10-fold higher in ALP activity respectively as compared to those treated with RA or the induction medium alone. RT-PCR analysis showed that such a synergistic effect of RA and the induction medium paralleled the process of inhibition on adipogenesis. Additional experiments showed that IBMX played a key role in increasing the effect of RA and ALP activity. Our results suggested that the relationship between adipogenesis and osteogenesis in ST2 cells was reciprocally interrelated and the process of adipogenesis could be potentially reversed into an osteoblastogenic tendency. This is the first report demonstrating that RA transforms adipogenic potential into an osteoblastic tendency.

  3. Cysteamine treatment restores the in vitro ability to differentiate along the osteoblastic lineage of mesenchymal stromal cells isolated from bone marrow of a cystinotic patient.

    PubMed

    Conforti, Antonella; Taranta, Anna; Biagini, Simone; Starc, Nadia; Pitisci, Angela; Bellomo, Francesco; Cirillo, Valentina; Locatelli, Franco; Bernardo, Maria Ester; Emma, Francesco

    2015-05-07

    Cystinosis is a rare autosomal recessive disease caused by mutations of the CTNS gene, which encodes for a lysosomal cystine/H(+) symporter. In mice, inactivation of the CTNS gene causes intralysosomal cystine accumulation and progressive organ damage that can be reversed, at least in part, by infusion of mesenchymal stromal cells (MSCs). Little is known on the mesenchymal compartment of cystinotic patients. The aim of the study was to test the phenotypical and functional properties of cystinotic MSCs (Cys-MSCs) isolated from bone marrow (BM) aspirate of a patient with nephropathic cystinosis. Morphology, proliferative capacity (measured as population doublings), immunophenotype (by flow-cytometry) and immunomodulatory properties (as phytohemagglutinin-induced peripheral blood mononuclear cell proliferation) were analyzed. The osteogenic differentiation potential of Cys-MSCs was evaluated by histological staining (alkaline phosphatase activity, Alzarin Red and von Kossa staining) spectrophotometry and Quantitative Reverse Transcriptase Polymerase Chain Reaction for osteigenic markers in the presence and in the absence of cysteamine. Cys-MSCs were compared with those isolated and expanded ex vivo from three healthy donors (HD-MSCs). Despite a slightly lower proliferative capacity, Cys-MSCs displayed a characteristic spindle-shaped morphology and similar immunephenotype as HD-MSCs. Cys-MSCs and HD-MSCs prevented proliferation of PHA-stimulated allogeneic peripheral blood mononuclear cells to the same extent. After in vitro induction into osteoblasts, Cys-MSCs showed reduced alkaline phosphatase (ALP) activity, calcium depositions and expression of ALP and collagen type 1. When Cys-MSCs were treated in vitro with increasing doses of cysteamine (50-100-200 μM/L) during the differentiation assay, recovery of Cys-MSCs differentiation capacity into osteoblasts was observed. No difference in adipogenic differentiation was found between Cys-MSCs and HD-MSCs. Our results

  4. Effects of transforming growth factor beta1 released from biodegradable polymer microparticles on marrow stromal osteoblasts cultured on poly(propylene fumarate) substrates.

    PubMed

    Peter, S J; Lu, L; Kim, D J; Stamatas, G N; Miller, M J; Yaszemski, M J; Mikos, A G

    2000-06-05

    Recombinant human transforming growth factor beta1 (TGF-beta1) was incorporated into microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) to create a delivery vehicle for the growth factor. The entrapment efficiency of TGF-beta1 in the microparticles containing 5% PEG was 40.3 +/- 1.2% for a TGF-beta1 loading density of 6.0 ng/1 mg of microparticles. For the same loading, 17.9 +/- 0.6 and 32.1 +/- 2.5% of the loaded TGF-beta1 was released after 1 and 8 days, respectively, followed by a plateau for the remaining 3 weeks. Rat marrow stromal cells showed a dose response to TGF-beta1 released from the microparticles similar to that of added TGF-beta1, indicating the activity of TGF-beta1 was retained during microparticle fabrication and after TGF-beta1 release. An optimal TGF-beta1 dosage of 1.0 ng/mL was determined through a 3-day dose response study for maximal alkaline phosphatase (ALP) activity. The TGF-beta1 released from the microparticles loaded with 6.0 ng TGF-beta1/1 mg of microparticles for the optimal dosage of TGF-beta1 enhanced the proliferation and osteoblastic differentiation of marrow stromal cells cultured on poly(propylene fumarate) substrates. The cells showed significantly increased total cell number, ALP activity, and osteocalcin production with values reaching 138,700 +/- 3300 cells/cm(2), 22.8 +/- 1.5 x 10(-7) micromol/min/cell, and 15.9 +/- 1.5 x 10(-6) ng/cell, respectively, after 21 days as compared to cells cultured under control conditions without TGF-beta1. These results suggest that controlled release of TGF-beta1 from the PLGA/PEG blend microparticles may find applications in modulating cellular response during bone healing at a skeletal defect site.

  5. Ectopic bone formation by marrow stromal osteoblast transplantation using poly(DL-lactic-co-glycolic acid) foams implanted into the rat mesentery

    NASA Technical Reports Server (NTRS)

    Ishaug-Riley, S. L.; Crane, G. M.; Gurlek, A.; Miller, M. J.; Yasko, A. W.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    1997-01-01

    Porous biodegradable poly(DL-lactic-co-glycolic acid) foams were seeded with rat marrow stromal cells and implanted into the rat mesentery to investigate in vivo bone formation at an ectopic site. Cells were seeded at a density of 6.83 x 10(5) cells/cm2 onto polymer foams having pore sizes ranging from either 150 to 300 to 710 microns and cultured for 7 days in vitro prior to implantation. The polymer/cell constructs were harvested after 1, 7, 28, or 49 days in vivo and processed for histology and gel permeation chromatography. Visual observation of hematoxylin and eosin-stained sections and von Kossa-stained sections revealed the formation of mineralized bonelike tissue in the constructs within 7 days postimplantation. Ingrowth of vascular tissue was also found adjacent to the islands of bone, supplying the necessary metabolic requirements to the newly formed tissue. Mineralization and bone tissue formation were investigated by histomorphometry. The average penetration depth of mineralized tissue in the construct ranged from 190 +/- 50 microns for foams with 500-710-microns pores to 370 +/- 160 microns for foams with 150-300-microns pores after 49 days in vivo. The mineralized bone volume per surface area and total bone volume per surface area had maximal values of 0.28 +/- 0.21 mm (500-710-microns pore size, day 28) and 0.038 +/- 0.024 mm (150-300-microns, day 28), respectively. As much as 11% of the foam volume penetrated by bone tissue was filled with mineralized tissue. No significant trends over time were observed for any of the measured values (penetration depth, bone volume/surface area, or percent mineralized bone volume). These results suggest the feasibility of bone formation by osteoblast transplantation in an orthotopic site where not only bone formation from transplanted cells but also ingrowth from adjacent bone may occur.

  6. Ectopic bone formation by marrow stromal osteoblast transplantation using poly(DL-lactic-co-glycolic acid) foams implanted into the rat mesentery.

    PubMed

    Ishaug-Riley, S L; Crane, G M; Gurlek, A; Miller, M J; Yasko, A W; Yaszemski, M J; Mikos, A G

    1997-07-01

    Porous biodegradable poly(DL-lactic-co-glycolic acid) foams were seeded with rat marrow stromal cells and implanted into the rat mesentery to investigate in vivo bone formation at an ectopic site. Cells were seeded at a density of 6.83 x 10(5) cells/cm2 onto polymer foams having pore sizes ranging from either 150 to 300 to 710 microns and cultured for 7 days in vitro prior to implantation. The polymer/cell constructs were harvested after 1, 7, 28, or 49 days in vivo and processed for histology and gel permeation chromatography. Visual observation of hematoxylin and eosin-stained sections and von Kossa-stained sections revealed the formation of mineralized bonelike tissue in the constructs within 7 days postimplantation. Ingrowth of vascular tissue was also found adjacent to the islands of bone, supplying the necessary metabolic requirements to the newly formed tissue. Mineralization and bone tissue formation were investigated by histomorphometry. The average penetration depth of mineralized tissue in the construct ranged from 190 +/- 50 microns for foams with 500-710-microns pores to 370 +/- 160 microns for foams with 150-300-microns pores after 49 days in vivo. The mineralized bone volume per surface area and total bone volume per surface area had maximal values of 0.28 +/- 0.21 mm (500-710-microns pore size, day 28) and 0.038 +/- 0.024 mm (150-300-microns, day 28), respectively. As much as 11% of the foam volume penetrated by bone tissue was filled with mineralized tissue. No significant trends over time were observed for any of the measured values (penetration depth, bone volume/surface area, or percent mineralized bone volume). These results suggest the feasibility of bone formation by osteoblast transplantation in an orthotopic site where not only bone formation from transplanted cells but also ingrowth from adjacent bone may occur.

  7. Upregulation of long non-coding RNA HIF 1α-anti-sense 1 induced by transforming growth factor-β-mediated targeting of sirtuin 1 promotes osteoblastic differentiation of human bone marrow stromal cells

    PubMed Central

    XU, YAO; WANG, SHILONG; TANG, CHAOLIANG; CHEN, WENJUN

    2015-01-01

    The present study aimed to investigate the regulatory mechanism of long non-coding RNA hypoxia-inducible factor 1α-anti-sense 1 (lncRNA HIF1α-AS1) in osteoblast differentiation as well as its targeting by sirtuin 1 (SIRT1), which may be inhibited by transforming growth factor (TGF)-β in bone marrow stromal cells (BMSCs). Real-time polymerase chain reaction (PCR), western blot analysis, lncRNA PCR arrays and chromatin immunoprecipitation were performed in order to examine the interference of SIRT1 expression by TGF-β, the effects of SIRT1 overexpression on lncRNA HIF1α-AS1 and the regulation of the expression of homeobox (HOX)D10, which promotes BMSC differentiation, by lncRNA HIF1α-AS1. The results showed that TGF-β interfered with SIRT1 expression. Furthermore, lncRNA HIF1α-AS1 was significantly downregulated following overexpression of SIRT1. In addition, low expression of HIF1α-AS1 was sufficient to block the expression of HOXD10. The present study further demonstrated that downregulation of HOXD10 by HIF1α-AS1 interfered with acetylation, and subsequently resulted in the inhibition of osteoblast differentiation. These results suggested that HIF1α-AS1 is an essential mediator of osteoblast differentiation, and may thus represent a gene-therapeutic agent for the treatment of human bone diseases. PMID:26460121

  8. Bone marrow osteoblast vulnerability to chemotherapy.

    PubMed

    Gencheva, Marieta; Hare, Ian; Kurian, Susan; Fortney, Jim; Piktel, Debbie; Wysolmerski, Robert; Gibson, Laura F

    2013-06-01

    Osteoblasts are a major component of the bone marrow microenvironment, which provide support for hematopoietic cell development. Functional disruption of any element of the bone marrow niche, including osteoblasts, can potentially impair hematopoiesis. We have studied the effect of two widely used drugs with different mechanisms of action, etoposide (VP16) and melphalan, on murine osteoblasts at distinct stages of maturation. VP16 and melphalan delayed maturation of preosteoblasts and altered CXCL12 protein levels, a key regulator of hematopoietic cell homing to the bone marrow. Sublethal concentrations of VP16 and melphalan also decreased the levels of several transcripts which contribute to the composition of the extracellular matrix (ECM) including osteopontin (OPN), osteocalcin (OCN), and collagen 1A1 (Col1a1). The impact of chemotherapy on message and protein levels for some targets was not always aligned, suggesting differential responses at the transcription and translation or protein stability levels. As one of the main functions of a mature osteoblast is to synthesize ECM of a defined composition, disruption of the ratio of its components may be one mechanism by which chemotherapy affects the ability of osteoblasts to support hematopoietic recovery coincident with altered marrow architecture. Collectively, these observations suggest that the osteoblast compartment of the marrow hematopoietic niche is vulnerable to functional dysregulation by damage imposed by agents frequently used in clinical settings. Understanding the mechanistic underpinning of chemotherapy-induced changes on the hematopoietic support capacity of the marrow microenvironment may contribute to improved strategies to optimize patient recovery post-transplantation.

  9. Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 3. Proliferation and differentiation of encapsulated marrow stromal osteoblasts cultured on crosslinking poly(propylene fumarate).

    PubMed

    Payne, Richard G; McGonigle, Joseph S; Yaszemski, Michael J; Yasko, Alan W; Mikos, Antonios G

    2002-11-01

    This study investigated the effect of temporary encapsulation of rat marrow stromal osteoblasts in crosslinked gelatin microparticles on long-term cell proliferation and phenotypic expression for microparticles placed on crosslinking poly(propylene fumarate) (PPF) composites using N-vinyl pyrollidinone (N-VP) as a crosslinking agent over a 28 day time period. Encapsulated cells (ECs) were seeded on actively crosslinking PPF composites 6 min after initiation of the crosslinking reaction, and also on fully crosslinked PPF composites and tissue culture polystyrene controls, with a cell seeding density of 5.3 x 10(4) cells/cm2. Composites prepared with three PPF:N-VP ratios were examined: 1:0.5, 1:0.1, and 1:0.05. Samples were taken at specified time points and analyzed by DNA, 3H-thymidine, alkaline phosphatase, osteocalcin, and calcium assays, and the measurements were compared with those for nonencapsulated cells (NCs). The results showed that encapsulated marrow stromal cells exhibited much higher viability, proliferation, and phenotypic expression when placed on crosslinking PPF composites than NCs. The assay results for ECs on crosslinking PPF composites were also similar to those on fully crosslinked PPF composites. The data further demonstrated that the PPF:N-VP ratio had no effect on the viability, proliferation, or phenotypic expression of the ECs. These results suggest that cells encapsulated in crosslinked gelatin microparticles may be part of an injectable, in situ crosslinkable, biodegradable polymeric composite for bone tissue engineering applications.

  10. Nicotine Increases Osteoblast Activity of Induced Bone Marrow Stromal Cells in a Dose-Dependent Manner: An in vitro Cell Culture Experiment.

    PubMed

    Daffner, Scott D; Waugh, Stacey; Norman, Timothy L; Mukherjee, Nilay; France, John C

    2012-09-01

    Previous studies by our group showed that nicotine delivered via a transdermal nicotine patch significantly enhanced posterior spinal fusion rates in rabbits. Nicotine transdermal patches provide a steady serum level; there may be a dose-dependent effect of nicotine on posterior spinal fusion. In an in vitro cell culture model of rabbit bone marrow-derived osteoblast-like cells, cells were exposed to different concentrations of nicotine (0, 20, 40, 80 ng/mL and 10, 100, 250 μg/mL). Wells were stained with an alkaline phosphatase (ALP) staining kit to determine ALP enzyme activity. Cells were stained with Von Kossa for mineralization. A two-way analysis of variance (ANOVA) using dose and time as variables showed significant differences among groups; post hoc analysis showed that the 100-μg/mL dose of nicotine significantly enhanced ALP activity over controls. A one-way ANOVA using dose as the variable showed that the 100- and 250-μg/mL doses had significantly greater mineralization than controls. Dose-response analysis revealed a statistically significant effect of nicotine dose on ALP activity and Von Kossa activity. The effects of nicotine on spinal fusion may be dose-dependent and due to stimulation of osteoblastic activity. Nicotine may not be responsible for the inhibited bone healing observed in smokers.

  11. Question of bone marrow stromal fibroblast traffic

    SciTech Connect

    Maloney, M.A.; Lamela, R.A.; Patt, H.M.

    1985-01-01

    Bone marrow stromal fibroblasts (CFU-F) normally do not exchange bone marrow sites in vivo. Restitution of the CFU-F after radiation damage is primarily recovery by the local fibroblasts from potentially lethal damage. Migration of stromal fibroblasts from shielded sites to an irradiated site makes a minimal contribution, if any, to CFU-F recovery. Determination of the relative contribution of donor stromal cells in bone marrow transplants by karyotyping the proliferating bone marrow stromal cells in vitro may not reflect the relative distribution of fibroblasts in the marrow. If there is residual damage to the host stromal fibroblasts from treatment before transplantation, these cells may not be able to proliferate in vitro. Therefore, an occasional transplanted fibroblast may contribute most of the metaphase figures scored for karyotype.

  12. Osteogenic effects of D+beta-3,4-dihydroxyphenyl lactic acid (salvianic acid A, SAA) on osteoblasts and bone marrow stromal cells of intact and prednisone-treated rats.

    PubMed

    Cui, Liao; Liu, Yu-Yu; Wu, Tie; Ai, Chun-Mei; Chen, Huai-Qing

    2009-03-01

    Previous studies have shown that D(+)beta-3,4-dihydroxyphenyl lactic acid (salvianic acid A, SAA) has anabolic effects on prednisone (GC)-induced osteoporosis in rats. The current study aims to investigate the molecular mechanism of SAA's impact on osteogenesis and adipogenesis in bone marrow stromal cells in intact and GC-treated rats. For in vitro study, newborn rat calvaria osteoblasts (rOBs) and rat bone marrow stromal cells (rMSCs) were isolated, identified and cultured with SAA at different concentrations to evaluate SAA's influence on osteogenesis and adipogenesis. In addition, 3-month-old Sprague-Dawley (SD) male rats were treated with distilled water, prednisone alone (3.0 mgxkg(-1)xd(-1)) or prednisone (3.0 mgxkg(-1)xd(-1)) and SAA (25 mgxkg(-1)xd(-1)) for 45 d. At the end point, the different groups of rMSCs were isolated by density-gradient centrifugation and cultured. (1) At 0.1-10.0 mg/L, SAA increased ALP activity, type I collagen (Coll-I) mRNA and OPG mRNA expression and stimulated nodule mineralization of rOBs. SAA (0.5 mg/L) also significantly increased the ALP activity of rMSCs without a need for osteogenesis-inducing medium. At 5.0 mg/L, SAA decreased the number of adipocytes with less lipid droplet formation from the rMSCs, which typically undergo adipocyte induction. (2) Coll-I expression was markedly decreased, whereas lipoprotein lipase (LPL) mRNA expression increased by 98% when compared with the first generation of rMSCs in GC-treated rats. The SAA-treated rats demonstrated an over 2-fold increase in Coll-I expression when compared with intact rats and further showed a significant decrease in LPL expression when compared with GC-treated rats. When rMSCs were co-cultured with SAA (0.5 mg/L) in vitro, SAA did not affect Coll-I and LPL gene expression in intact rats but significantly increased Coll-I and decreased LPL gene expression in GC-treated rats. SAA protected bone from GC-induced bone marrow impairment by stimulating osteogenesis and

  13. Osteogenic effects of D(+)β-3,4-dihydroxyphenyl lactic acid (salvianic acid A, SAA) on osteoblasts and bone marrow stromal cells of intact and prednisone-treated rats

    PubMed Central

    Cui, Liao; Liu, Yu-yu; Wu, Tie; Ai, Chun-mei; Chen, Huai-qing

    2009-01-01

    Aim: Previous studies have shown that D(+)β-3,4-dihydroxyphenyl lactic acid (salvianic acid A, SAA) has anabolic effects on prednisone (GC)-induced osteoporosis in rats. The current study aims to investigate the molecular mechanism of SAA's impact on osteogenesis and adipogenesis in bone marrow stromal cells in intact and GC-treated rats. Methods: For in vitro study, newborn rat calvaria osteoblasts (rOBs) and rat bone marrow stromal cells (rMSCs) were isolated, identified and cultured with SAA at different concentrations to evaluate SAA's influence on osteogenesis and adipogenesis. In addition, 3-month-old Sprague-Dawley (SD) male rats were treated with distilled water, prednisone alone (3.0 mg·kg−1·d−1) or prednisone (3.0 mg·kg−1·d−1) and SAA (25 mg·kg−1·d−1) for 45 d. At the end point, the different groups of rMSCs were isolated by density-gradient centrifugation and cultured. Results: (1) At 0.1–10.0 mg/L, SAA increased ALP activity, type I collagen (Coll-I) mRNA and OPG mRNA expression and stimulated nodule mineralization of rOBs. SAA (0.5 mg/L) also significantly increased the ALP activity of rMSCs without a need for osteogenesis-inducing medium. At 5.0 mg/L, SAA decreased the number of adipocytes with less lipid droplet formation from the rMSCs, which typically undergo adipocyte induction. (2) Coll-I expression was markedly decreased, whereas lipoprotein lipase (LPL) mRNA expression increased by 98% when compared with the first generation of rMSCs in GC-treated rats. The SAA-treated rats demonstrated an over 2-fold increase in Coll-I expression when compared with intact rats and further showed a significant decrease in LPL expression when compared with GC-treated rats. When rMSCs were co-cultured with SAA (0.5 mg/L) in vitro, SAA did not affect Coll-I and LPL gene expression in intact rats but significantly increased Coll-I and decreased LPL gene expression in GC-treated rats. Conclusion: SAA protected bone from GC-induced bone marrow

  14. Bone marrow osteoblast vulnerability to chemotherapy

    PubMed Central

    Gencheva, Marieta; Hare, Ian; Kurian, Susan; Fortney, Jim; Piktel, Debbie; Wysolmerski, Robert; Gibson, Laura F.

    2013-01-01

    Osteoblasts are a major component of the bone marrow microenvironment which provide support for hematopoietic cell development. Functional disruption of any element of the bone marrow niche, including osteoblasts, can potentially impair hematopoiesis. We have studied the effect of two widely used drugs with different mechanisms of action, etoposide (VP16) and melphalan, on murine osteoblasts at distinct stages of maturation. VP16 and melphalan delayed maturation of preosteoblasts and altered CXCL12 protein levels, a key regulator of hematopoietic cell homing to the bone marrow. Sublethal concentrations of VP16 and melphalan also decreased the levels of several transcripts which contribute to the composition of the extracellular matrix (ECM) including osteopontin (OPN), osteocalcin (OCN) and collagen 1A1 (Col1a1). The impact of chemotherapy on message and protein levels for some targets was not always aligned, suggesting differential responses at the transcription and translation or protein stability levels. Since one of the main functions of a mature osteoblast is to synthesize ECM of a defined composition, disruption of the ratio of its components may be one mechanism by which chemotherapy affects the ability of osteoblasts to support hematopoietic recovery coincident with altered marrow architecture. Collectively, these observations suggest that the osteoblast compartment of the marrow hematopoietic niche is vulnerable to functional dysregulation by damage imposed by agents frequently used in clinical settings. Understanding the mechanistic underpinning of chemotherapy-induced changes on the hematopoietic support capacity of the marrow microenvironment may contribute to improved strategies to optimize patient recovery post-transplantation. PMID:23551534

  15. Construction of microenvironment onto titanium substrates to regulate the osteoblastic differentiation of bone marrow stromal cells in vitro and osteogenesis in vivo.

    PubMed

    Lai, Min; Cai, Kaiyong; Hu, Yan; Zhang, Yuan; Li, Liqi; Luo, Zhong; Hou, Yanhua; Li, Jinghua; Ding, Xingwei; Chen, Xiuyong

    2013-03-01

    To mimic the extracellular matrix of natural bone, apatite/gelatin composite was deposited onto nanostructured titanium substrates via a coprecipitation method, which was pretreated by potassium hydroxide and heat treatment to generate an anticorrosive nanostructured layer. The successful formation of the apatite/gelatin nanocomposite onto titanium surfaces was revealed by Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, atomic force microscopy (AFM), and thin film X-ray diffraction (TF-XRD) measurements, respectively. The immunofluorescence staining of vinculin revealed that the apatite/gelatin nanocomposite deposited titanium substrate was favorable for cell adhesion. More importantly, bone marrow stromal cells cultured onto the apatite/gelatin nanocomposite deposited titanium substrates displayed significantly higher (p < 0.05 or p < 0.01) proliferation and differentiation levels of alkaline phosphatase, mRNA expressions of osteocalcin (OC), osteopontin (OPN), and collagen type I (Col I), and OC content after culture for 7, 14, and 21 days, respectively, which was also revealed by the immunofluorescence analysis of OC and OPN expression. The deposition of apatite/gelatin nanocomposite improved bone density (p < 0.05) and bone-implant contact rate (p < 0.05), which was reflected by microcomputed tomography analysis and histological evaluation in vivo using a rabbit model. This work provides an approach to fabricate high-performance titanium-based implants with enhanced bone osseointegration.

  16. Modulation of marrow stromal osteoblast adhesion on biomimetic oligo[poly(ethylene glycol) fumarate] hydrogels modified with Arg-Gly-Asp peptides and a poly(ethyleneglycol) spacer.

    PubMed

    Shin, Heungsoo; Jo, Seongbong; Mikos, Antonios G

    2002-08-01

    Novel oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels functionalized with cell adhesion peptides were prepared, and the effects of incorporated peptide density and macromolecular structure of hydrogels on attachment and morphology of marrow stromal cells (MSCs) were evaluated. Poly(ethylene glycol) (PEG; number average molecular weight of 930, 2860, and 6090) was used to synthesize OPF. A model peptide, Gly-Arg-Gly-Asp (GRGD), was incorporated into OPF hydrogels after being coupled to acrylated PEG of molecular weight 3400. The increase of incorporated peptide concentration enhanced MSC attachment to OPF hydrogels of PEG of molecular weight of 930 and 2860. However, the number of attached MSCs to OPF hydrogels of PEG (molecular weight 6090) remained constant regardless of the peptide density. The length of PEG in OPF also influenced cell attachment. When 1 micromole peptide/g hydrogel was incorporated into the OPF hydrogels, the degree of cell attachment at 12 h relative to the initial seeding density was 93.9 +/- 5.9%, 64.7 +/- 8.2%, and 9.3 +/- 6.6% for OPF hydrogels prepared with PEG of molecular weights of 930, 2860, and 6090, respectively. However, the crosslinking density of hydrogels did not significantly affect cell attachment. The interaction was sequence specific, in that MSC attachment to GRGD-modified hydrogels was competitively inhibited when cells were incubated in the presence of 0.5 mM soluble GRGD before cell seeding. These results suggest that we can modulate MSC attachment to OPF hydrogels by altering the peptide density and the molecular structure of OPF hydrogels.

  17. Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 2. Viability of encapsulated marrow stromal osteoblasts cultured on crosslinking poly(propylene fumarate).

    PubMed

    Payne, Richard G; McGonigle, Joseph S; Yaszemski, Michael J; Yasko, Alan W; Mikos, Antonios G

    2002-11-01

    The effect of temporary encapsulation of rat marrow stromal osteoblasts in crosslinked gelatin microparticles on cell viability and proliferation was investigated in this study for microparticles placed on a crosslinking poly(propylene fumarate) (PPF) composite over a 7 day time period. Encapsulated cells were seeded on crosslinking PPF composites at times up to 10 min following initiation of the crosslinking reaction, and also on fully crosslinked PPF composites and tissue culture polystyrene controls, with a cell seeding density of 5.3 x 10(4) cells/cm2. The crosslinked PPF composite exhibited an average gel point of 10.3 min and an average maximum crosslinking temperature of 47.5 degrees C. Cell viability and proliferation were assessed by DNA and 3H-thymidine assays and the results were compared with those for nonencapsulated cells. The results showed that the addition time of cells to a crosslinking PPF composite had a large effect on cell viability and proliferation for both encapsulated and nonencapsulated cells with more surviving cells added at later time points. Most importantly, the temporary encapsulation of cells significantly enhanced cell viability at earlier time points. The data indicate that the presence of gelatin microparticles does not affect the crosslinking of a PPF composite. They further suggest that the temporary encapsulation of cells in crosslinked gelatin microparticles may preserve the viability of cells contained in an actively crosslinking PPF composite used as an injectable polymeric scaffold serving also as a carrier for osteogenic cell populations.

  18. The effect of autologous bone marrow stromal cells differentiated on scaffolds for canine tibial bone reconstruction.

    PubMed

    Özdal-Kurt, F; Tuğlu, I; Vatansever, H S; Tong, S; Deliloğlu-Gürhan, S I

    2015-01-01

    Bone marrow contains mesenchymal stem cells that form many tissues. Various scaffolds are available for bone reconstruction by tissue engineering. Osteoblastic differentiated bone marrow stromal cells (BMSC) promote osteogenesis on scaffolds and stimulate bone regeneration. We investigated the use of cultured autologous BMSC on different scaffolds for healing defects in tibias of adult male canines. BMSC were isolated from canine humerus bone marrow, differentiated into osteoblasts in culture and loaded onto porous ceramic scaffolds including hydroxyapatite 1, hydroxyapatite gel and calcium phosphate. Osteoblast differentiation was verified by osteonectine and osteocalcine immunocytochemistry. The scaffolds with stromal cells were implanted in the tibial defect. Scaffolds without stromal cells were used as controls. Sections from the defects were processed for histological, ultrastructural, immunohistochemical and histomorphometric analyses to analyze the healing of the defects. BMSC were spread, allowed to proliferate and differentiate to osteoblasts as shown by alizarin red histochemistry, and osteocalcine and osteonectine immunostaining. Scanning electron microscopy showed that BMSC on the scaffolds were more active and adhesive to the calcium phosphate scaffold compared to the others. Macroscopic bone formation was observed in all groups, but scaffolds with stromal cells produced significantly better results. Bone healing occurred earlier and faster with stromal cells on the calcium phosphate scaffold and produced more callus compared to other scaffolds. Tissue healing and osteoblastic marker expression also were better with stromal cells on the scaffolds. Increased trabecula formation, cell density and decreased fibrosis were observed in the calcium phosphate scaffold with stromal cells. Autologous cultured stromal cells on the scaffolds were useful for healing of canine tibial bone defects. The calcium phosphate scaffold was the best for both cell

  19. Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development.

    PubMed

    Mizoguchi, Toshihide; Pinho, Sandra; Ahmed, Jalal; Kunisaki, Yuya; Hanoun, Maher; Mendelson, Avital; Ono, Noriaki; Kronenberg, Henry M; Frenette, Paul S

    2014-05-12

    Mesenchymal stem and progenitor cells (MSPCs) contribute to bone marrow (BM) homeostasis by generating multiple types of stromal cells. MSPCs can be labeled in the adult BM by Nestin-GFP, whereas committed osteoblast progenitors are marked by Osterix expression. However, the developmental origin and hierarchical relationship of stromal cells remain largely unknown. Here, by using a lineage-tracing system, we describe three distinct waves of contributions of Osterix(+) cells in the BM. First, Osterix(+) progenitors in the fetal BM contribute to nascent bone tissues and transient stromal cells that are replaced in the adult marrow. Second, Osterix-expressing cells perinatally contribute to osteolineages and long-lived BM stroma, which have characteristics of Nestin-GFP(+) MSPCs. Third, Osterix labeling in the adult marrow is osteolineage-restricted, devoid of stromal contribution. These results uncover a broad expression profile of Osterix and raise the intriguing possibility that distinct waves of stromal cells, primitive and definitive, may organize the developing BM.

  20. COL1A1 and miR-29b show lower expression levels during osteoblast differentiation of bone marrow stromal cells from Osteogenesis Imperfecta patients

    PubMed Central

    2014-01-01

    Background The majority of Osteogenesis Imperfecta (OI) cases are caused by mutations in one of the two genes, COL1A1 and COL1A2 encoding for the two chains that trimerize to form the procollagen 1 molecule. However, alterations in gene expression and microRNAs (miRNAs) are responsible for the regulation of cell fate determination and may be evolved in OI phenotype. Methods In this work, we analyzed the coding region and intron/exon boundaries of COL1A1 and COL1A2 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. COL1A1 and miR-29b expression were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System. Results We have identified eight novel mutations, where of four may be responsible for OI phenotype. COL1A1 and miR-29b showed lower expression values in OI type I and type III samples. Interestingly, one type III OI sample from a patient with Bruck Syndrome showed COL1A1 and miR-29b expressions alike those from normal samples. Conclusions Results suggest that the miR-29b mechanism directed to regulate collagen protein accumulation during mineralization is dependent upon the amount of COL1A1 mRNA. Taken together, results indicate that the lower levels observed in OI samples were not sufficient for the induction of miR-29b. PMID:24767406

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

    SciTech Connect

    Tu Qisheng; Valverde, Paloma . E-mail: paloma.valverde@tufts.edu; Chen, Jake

    2006-03-24

    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 {alpha}1(II) procollagen or lipoprotein lipase, which are markers of chondrogenic and adipogenic differentiation, respectively.

  2. Schwann cells induce neuronal differentiation of bone marrow stromal cells.

    PubMed

    Zurita, Mercedes; Vaquero, Jesús; Oya, Santiago; Miguel, Miriam

    2005-04-04

    Bone marrow stromal cells are multipotent stem cells that have the potential to differentiate into bone, cartilage, fat and muscle. Recently, bone marrow stromal cells have been shown to have the capacity to differentiate into neurons under specific experimental conditions, using chemical factors. We now describe how bone marrow stromal cells can be induced to differentiate into neuron-like cells when they are co-cultured with Schwann cells. When compared with chemical differentiation, expression of neuronal differentiation markers begins later, but one week after beginning co-culture, most bone marrow stromal cells showed a typical neuronal morphology. Our present findings support the transdifferentiation of bone marrow stromal cells, and the potential utility of these cells for the treatment of degenerative and acquired disorders of the nervous system.

  3. Asiatic acid inhibits adipogenic differentiation of bone marrow stromal cells.

    PubMed

    Li, Zheng-Wei; Piao, Cheng-dong; Sun, Hong-hui; Ren, Xian-Sheng; Bai, Yun-Shen

    2014-03-01

    Bone marrow mesenchymal stromal cells (BMSCs) are the common precursors for both osteoblasts and adipocytes. With aging, BMSC osteoblast differentiation decreases whereas BMSC differentiation into adipocytes increases, resulting in increased adipogenesis and bone loss. In the present study, we investigated the effect of asiatic acid (AA) on adipocytic differentiation of BMSCs. AA inhibited the adipogenic induction of lipid accumulation, activity of glycerol-3-phosphate dehydrogenase, and expression of marker genes in adipogenesis: peroxisome proliferation-activated receptor (PPAR)γ, adipocyte fatty acid-binding protein (ap) 2, and adipsin. Further, we found that AA did not alter clonal expansion rate and expression of C/EBPβ, upstream key regulator of PPARγ, and binding activity of C/EBPβ to PPARγ promoter was not affected by AA as well. These findings suggest that AA may modulate differentiation of BMSCs to cause a lineage shift away from the adipocytes, and inhibition of PPARγ by AA is through C/EBPβ-independent mechanisms. Thus, AA could be a potential candidate for a novel drug against osteoporosis.

  4. Osteogenic Potential of Mandibular vs. Long-bone Marrow Stromal Cells

    PubMed Central

    Aghaloo, T.L.; Chaichanasakul, T.; Bezouglaia, O.; Kang, B.; Franco, R.; Dry, S.M.; Atti, E.; Tetradis, S.

    2010-01-01

    Although fundamentally similar to other bones, the jaws demonstrate discrete responses to developmental, mechanical, and homeostatic regulatory signals. Here, we hypothesized that rat mandible vs. long-bone marrow-derived cells possess different osteogenic potential. We established a protocol for rat mandible and long-bone marrow stromal cell (BMSC) isolation and culture. Mandible BMSC cultures formed more colonies, suggesting an increased CFU-F population. Both mandible and long-bone BMSCs differentiated into osteoblasts. However, mandible BMSCs demonstrated augmented alkaline phosphatase activity, mineralization, and osteoblast gene expression. Importantly, upon implantation into nude mice, mandible BMSCs formed 70% larger bone nodules containing three-fold more mineralized bone compared with long-bone BMSCs. Analysis of these data demonstrates an increased osteogenic potential and augmented capacity of mandible BMSCs to induce bone formation in vitro and in vivo. Our findings support differences in the mechanisms underlying mandible homeostasis and the pathophysiology of diseases unique to the jaws. PMID:20811069

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

    PubMed Central

    Tencerova, Michaela; Kassem, Moustapha

    2016-01-01

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

  6. Fibroblast Growth Factor-2 facilitates the growth and chemo-resistance of leukemia cells in the bone marrow by modulating osteoblast functions

    PubMed Central

    Sugimoto, Keiki; Miyata, Yasuhiko; Nakayama, Takayuki; Saito, Shigeki; Suzuki, Ritsuro; Hayakawa, Fumihiko; Nishiwaki, Satoshi; Mizuno, Hiroki; Takeshita, Kyosuke; Kato, Hidefumi; Ueda, Ryuzo; Takami, Akiyoshi; Naoe, Tomoki

    2016-01-01

    Stromal cells and osteoblasts play major roles in forming and modulating the bone marrow (BM) hematopoietic microenvironment. We have reported that FGF2 compromises stromal cell support of normal hematopoiesis. Here, we examined the effects of FGF2 on the leukemia microenvironment. In vitro, FGF2 significantly decreased the number of stromal-dependent and stromal-independent G0-leukemia cells in the stromal layers. Accordingly, CML cells placed on FGF2-treated stromal layers were more sensitive to imatinib. Conversely, FGF2 increased the proliferation of osteoblasts via FGFR1 IIIc, but its effects on osteoblast support of leukemia cell growth were limited. We next treated a human leukemia mouse model with Ara-C with/without systemic FGF2 administration. BM sections from FGF2-treated mice had thickened bone trabeculae and increased numbers of leukemia cells compared to controls. Leukemia cell density was increased, especially in the endosteal region in FGF2/Ara-C -treated mice compared to mice treated with Ara-C only. Interestingly, FGF2 did not promote leukemia cell survival in Ara-C treated spleen. Microarray analysis showed that FGF2 did not alter expression of many genes linked to hematopoiesis in osteoblasts, but modulated regulatory networks involved in angiogenesis and osteoblastic differentiation. These observations suggest that FGF2 promotes leukemia cell growth in the BM by modulating osteoblast functions. PMID:27481339

  7. Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix.

    PubMed

    Gronthos, S; Simmons, P J; Graves, S E; Robey, P G

    2001-02-01

    marrow microenvironment and bone surfaces. Furthermore, these data suggest a possible role for the beta1 integrin subfamily during the development of stromal precursor cells into functional osteoblast-like cells.

  8. The bone marrow microenvironment contributes to type I diabetes induced osteoblast death.

    PubMed

    Coe, Lindsay M; Irwin, Regina; Lippner, Dennean; McCabe, Laura R

    2011-02-01

    Type I diabetes increases an individual's risk for bone loss and fracture, predominantly through suppression of osteoblast activity (bone formation). During diabetes onset, levels of blood glucose and pro-inflammatory cytokines (including tumor necrosis factor α (TNFα)) increased. At the same time, levels of osteoblast markers are rapidly decreased and stay decreased chronically (i.e., 40 days later) at which point bone loss is clearly evident. We hypothesized that early bone marrow inflammation can promote osteoblast death and hence reduced osteoblast markers. Indeed, examination of type I diabetic mouse bones demonstrates a greater than twofold increase in osteoblast TUNEL staining and increased expression of pro-apoptotic factors. Osteoblast death was amplified in both pharmacologic and spontaneous diabetic mouse models. Given the known signaling and inter-relationships between marrow cells and osteoblasts, we examined the role of diabetic marrow in causing the osteoblast death. Co-culture studies demonstrate that compared to control marrow cells, diabetic bone marrow cells increase osteoblast (MC3T3 and bone marrow derived) caspase 3 activity and the ratio of Bax/Bcl-2 expression. Mouse blood glucose levels positively correlated with bone marrow induced osteoblast death and negatively correlated with osteocalcin expression in bone, suggesting a relationship between type I diabetes, bone marrow and osteoblast death. TNF expression was elevated in diabetic marrow (but not co-cultured osteoblasts); therefore, we treated co-cultures with TNFα neutralizing antibodies. The antibody protected osteoblasts from bone marrow induced death. Taken together, our findings implicate the bone marrow microenvironment and TNFα in mediating osteoblast death and contributing to type I diabetic bone loss.

  9. URB expression in human bone marrow stromal cells and during mouse development.

    PubMed

    Liu, Yi; Monticone, Massimiliano; Tonachini, Laura; Mastrogiacomo, Maddalena; Marigo, Valeria; Cancedda, Ranieri; Castagnola, Patrizio

    2004-09-17

    Seven genes preferentially expressed in undifferentiated human bone marrow stromal cells (BMSC) with respect to BMSC-derived osteoblasts were previously identified by differential display. Here we characterize the expression of one of these genes, URB, belonging to the sushi-repeat-containing protein superfamily. In culture, URB is expressed in both human primary and cloned BMSC, and is drastically downregulated during osteoblastic differentiation of these cells. Here we show that in mouse tissues a single 3.8kb Urb transcript is detected and that the mouse Urb protein is secreted as a 150kDa glycoprotein. During mouse development Urb RNA is barely detectable in 9dpc embryos and increases at later stages. Both in situ hybridization and immunohistochemistry analysis show Urb expression in mouse embryos starting from 14dpc mostly in cartilage. The temporal and spatial expression pattern of Urb suggests its role in mouse skeletogenesis.

  10. Factors affecting mesenchymal stromal cells yield from bone marrow aspiration.

    PubMed

    Li, Jing; Wong, Wilfred Hing-Sang; Chan, Shing; Chim, James Chor-San; Cheung, Kenneth Man-Chee; Lee, Tsz-Leung; Au, Wing-Yan; Ha, Shau-Yin; Lie, Albert Kwok-Wei; Lau, Yu-Lung; Liang, Raymond Hin-Suen; Chan, Godfrey Chi-Fung

    2011-03-01

    This study was to investigate the variables in bone marrow harvesting procedure and individual donor factors which can potentially affect the yield of mesenchymal stromal cells (MSC). WE DETERMINED THE YIELD OF MSC FROM BONE MARROW UNDER DIFFERENT CLINICAL CONDITIONS BY COMPARING THE MSC COLONY NUMBERS FROM: (1) donors of different ages; (2) healthy donors and patients with leukemia; (3) bone marrow aspirated at different time points during marrow harvesting; (4) bone marrow harvested by different needles. During the process of harvesting, the number of MSC significantly decreased with increase number of aspiration, from 675/ml at the initial decreased to 60/ml after 100 ml bone marrow aspirated, and 50/ml after 200 ml bone marrow aspirated. The number of MSC retrieved from leukemia patients (99/ml bone marrow) was significantly lower than that of healthy donors (708/ml bone marrow). However, there was no significant difference in growth rate. There was no significant age-related difference of MSC yielded from donors <55 years. And there was no significant difference in MSC number between the samples from single end-holed needle and those from multiple-side-hole needle. The optimal bone marrow samples for MSC collection should be obtained earlier in the process of harvesting procedure. Bone marrow from donors <55 years was equally good as MSC sources. The autologous MSC from leukemia patients can be utilized for in-vitro MSC expansion.

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

    PubMed

    Jafari, Abbas; Qanie, Diyako; Andersen, Thomas L; Zhang, Yuxi; Chen, Li; Postert, Benno; Parsons, Stuart; Ditzel, Nicholas; Khosla, Sundeep; Johansen, Harald Thidemann; Kjærsgaard-Andersen, Per; Delaisse, Jean-Marie; Abdallah, Basem M; Hesselson, Daniel; Solberg, Rigmor; Kassem, Moustapha

    2017-02-14

    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.

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

    PubMed

    Meng, Jingru; Ma, Xue; Wang, Ning; Jia, Min; Bi, Long; Wang, Yunying; Li, Mingkai; Zhang, Huinan; Xue, Xiaoyan; Hou, Zheng; Zhou, Ying; Yu, Zhibin; He, Gonghao; Luo, Xiaoxing

    2016-04-12

    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.

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

    PubMed Central

    Meng, Jingru; Ma, Xue; Wang, Ning; Jia, Min; Bi, Long; Wang, Yunying; Li, Mingkai; Zhang, Huinan; Xue, Xiaoyan; Hou, Zheng; Zhou, Ying; Yu, Zhibin; He, Gonghao; Luo, Xiaoxing

    2016-01-01

    Summary 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. PMID:26947974

  14. Marrow stromal fibroblastic cell cultivation in vitro on decellularized bone marrow extracellular matrix.

    PubMed

    Dutra, Timothy F; French, Samuel W

    2010-02-01

    The in vitro biocompatibility of decellularized bone marrow extracellular matrix was evaluated. Following a freeze-thaw cycle, sectioned discs of fresh frozen rat metaphyseal bone were sequentially incubated in solutions of hypertonic, then hypotonic Ringer's solution, followed by deoxycholic acid, then DNAase I. The adequacy of decellularization of marrow stroma was examined by light microscopy. Marrow stromal fibroblastic cells were harvested by dispersion of rat long bone marrow, followed by concentration by discontinuous Ficoll-Paque gradient centrifugation. The fibroblastic cells were expanded by in vitro cultivation, and second passage cells were cryopreserved until needed. Cryopreserved marrow stromal cells were applied dropwise to sections of decellularized bone marrow extracellular matrix, and cultured in BJGb medium with 20% fetal bovine serum for ten days. Mature cultures were formalin fixed, decalcified, and embedded in paraffin. Light microscopy of hematoxylin and eosin stained sections showed individual spindle cells invading the upper portion of the decellularized extracellular matrix, and also a monolayer of spindle cells on the upper surfaces of exposed trabecular and cortical bone. This experiment showed that decellularized marrow extracellular matrix is a biocompatible three dimensional in vitro substrate for marrow stromal fibroblastic cells.

  15. Vitamin D Metabolism and Action in Human Bone Marrow Stromal Cells

    PubMed Central

    Zhou, Shuanhu; LeBoff, Meryl S.; Glowacki, Julie

    2010-01-01

    Vitamin D metabolites are important effectors of bone and mineral homeostasis. Extrarenal conversion of 25-hydroxyvitamin D (25OHD) to the biologically active form of vitamin D, 1α,25-dihydroxyvitamin D [1,25(OH)2D] is catalyzed in several cell types by the 1α-hydroxylase (CYP27B1), but little is known about the expression or regulation of CYP27B1 in human bones. We examined whether human bone marrow stromal cells (hMSCs, also known as mesenchymal stem cells) participate in vitamin D metabolism and whether vitamin D hydroxylases in hMSCs are influenced by the vitamin D status of the individual from whom the hMSCs were obtained. We also investigated the effects of vitamin D metabolites on osteoblast differentiation and the role of IGF-I in the regulation of CYP27B1. In a series of 27 subjects, vitamin D hydroxylases in hMSCs were expressed at different levels and were correlated with serum 25OHD, 1,25(OH)2D, and PTH. In vitro treatment with 25OHD up-regulated CYP27B1 and IGF-I in hMSCs; IGF-I also up-regulated CY27B1 expression and stimulated osteoblast differentiation. When hydroxylation of 25OHD was blocked by ketoconazole, a cytochrome P450 inhibitor, 25OHD was no longer able to induce CYP27B1 expression. In summary, these findings show that human bone marrow stromal cells have the molecular machinery both to metabolize and respond to vitamin D. We propose that circulating 25OHD, by virtue of its local conversion to 1,25(OH)2D catalyzed by basal CYP27B1 in hMSCs, amplifies vitamin D signaling through IGF-I up-regulation, which in turn induces CYP27B1 in a feed-forward mechanism to potentiate osteoblast differentiation initiated by IGF-I. PMID:19966181

  16. Effect of Coadministration of Vancomycin and BMP-2 on Cocultured Staphylococcus aureus and W-20-17 Mouse Bone Marrow Stromal Cells in Vitro

    DTIC Science & Technology

    2012-07-01

    Hudson MC. 2000. Intracellular Staph - ylococcus aureus induces apoptosis in mouse osteoblasts. FEMS Micro- biol. Lett. 186:151–156. 45. Wang Y, et al...Staphylococcus aureus and W-20-17 Mouse Bone Marrow Stromal Cells In Vitro 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S...Cocultured Staphylococcus aureus and W-20-17 Mouse Bone Marrow Stromal Cells In Vitro A. H. Nguyen,a S. Kim,a,b W. J. Maloney,b J. C. Wenke,c and Y. Yanga

  17. Confocal images of marrow stromal (Westen-Bainton) cells.

    PubMed

    Bianco, P; Boyde, A

    1993-08-01

    A cytochemical method was used for imaging a defined subset of marrow stromal cells (alkaline phosphatase-positive reticulum cells, hereinafter referred to as Westen-Bainton cells), which are endowed with membrane-associated alkaline phosphatase. The use of two different types of confocal microscopes was compared: a tandem scanning reflected light microscope and a laser scanning confocal microscope equipped with a 633 nm (helium-neon) laser. Sharp confocal reflection images of the cytochemically stained stromal cells were obtained with both microscopes. Three-dimensional reconstructions were generated with both systems, revealing morphological features of Westen-Bainton cells related to both their actual shape and organization within tissue architecture, which were not otherwise appreciated. The observations were extended to individual cases of bone pathology, and demonstrated the value of confocal microscopy for the investigation of marrow-bone relationships in physiology and disease.

  18. PRIMARY MARROW DERIVED STROMAL CELLS: ISOLATION AND MANIPULATION

    PubMed Central

    Ramakrishnan, Aravind; Torok-Storb, Beverly; Pillai, Manoj M

    2013-01-01

    Marrow Stromal Cells (MSCs) are relatively rare cells difficult to visualize in marrow biopsies or detect in aspirated marrow. Under specific conditions MSC can be expanded in vitro and the population can give rise to several mesenchymal lineages. “MSC” also refers to mesenchymal stem cells which implies that all cells in the population are multipotent. It is generally agreed that while there may be a few multipotent stem cells in an MSC population the majority are not stem cells. In either case MSC do not produce hematopoietic cells. Although MSCs have been isolated and characterized from several tissues, bone marrow is their most common source for research and clinical use. Primary MSC populations can be derived from bone marrow mononuclear cells with relative ease, but it is important to recognize the cellular heterogeneity within a culture and how this may vary from donor to donor. In this chapter, we will describe methodology to derive primary MSCs from bone marrow screens, an otherwise discarded byproduct of bone marrow harvests used for clinical transplantation. We will also describe some useful techniques to characterize and manipulate MSCs – both primary and immortalized cell lines. PMID:23959984

  19. Primary marrow-derived stromal cells: isolation and manipulation.

    PubMed

    Ramakrishnan, Aravind; Torok-Storb, Beverly; Pillai, Manoj M

    2013-01-01

    Marrow stromal cells (MSCs) are relatively rare cells difficult to visualize in marrow biopsies or detect in aspirated marrow. Under specific conditions MSC can be expanded in vitro and the population can give rise to several mesenchymal lineages. "MSC" also refers to mesenchymal stem cells which implies that all cells in the population are multipotent. It is generally agreed that while there may be a few multipotent stem cells in an MSC population the majority are not stem cells. In either case MSCs do not produce hematopoietic cells. Although MSCs have been isolated and characterized from several tissues, bone marrow is their most common source for research and clinical use. Primary MSC populations can be derived from bone marrow mononuclear cells with relative ease, but it is important to recognize the cellular heterogeneity within a culture and how this may vary from donor to donor. In this chapter, we describe methodology to derive primary MSCs from bone marrow screens, an otherwise discarded by-product of bone marrow harvests used for clinical transplantation. We also describe some useful techniques to characterize and manipulate MSCs-both primary and immortalized cell lines.

  20. Bone marrow-derived osteoblasts seeded into porous beta-tricalcium phosphate to repair segmental defect in canine's mandibula.

    PubMed

    Wu, Wei; Chen, Xiaobin; Mao, Tianqiu; Chen, Fulin; Feng, Xinghua

    2006-10-01

    Bone regeneration is often needed for many aesthetic and reconstructive procedures. Tissue engineering provided a promising approach to supplement existing treatment strategies. In this study, we aimed to evaluate the effect of reconstructing mandibular defect by using bioceramics seeded with bone marrow derived osteoblasts. Canine's autologous marrow stromal cells were Culture-expanded and induced to osteoblastic phenotype, then were seeded into prepared porous beta-tricalcium phosphate, after being incubated in vitro. The cell/ scaffold complexes were implanted into the prepared defect in canines' mandibula and fixed by internal rigid fixation. In control groups, beta-tricalcium phosphate alone and autologous iliums were implanted into the prepared defects. Twelve weeks after implantation, the specimens were examined macroscopically and histologically. In experimental group and autologous iliums group, new bone grafts were successfully developed at 12 weeks after implantation and repaired the continuity of the mandibula. Histologically, newly formed bone could be observed on the surface and in the pores of beta-tricalcium phosphate in the cell/scaffold group, whereas incomplete bone repair was found in pure beta-tricalcium phosphate group. The harvested bone marrow derived osteoblasts possess the ability to form new bone tissue when seeded onto porous beta-tricalcium phosphate, which shows the potential of using this method to repair large segmental mandibular defect clinically.

  1. Enhancement of osteoblastic differentiation of mesenchymal stromal cells cultured by selective combination of bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2).

    PubMed

    Maegawa, Naoki; Kawamura, Kenji; Hirose, Motohiro; Yajima, Hiroshi; Takakura, Yoshinori; Ohgushi, Hajime

    2007-01-01

    It is well known that bone marrow contains mesenchymal stromal cells (MSCs), which can show osteoblastic differentiation when cultured in osteogenic medium containing ascorbic acid, beta-glycerophosphate and dexamethasone. The differentiation results in the appearance of osteoblasts, together with the formation of bone matrix; thus, in vitro cultured bone (osteoblasts/bone matrix) could be fabricated by MSC culture. This type of cultured bone has already been used in clinical cases involving orthopaedic problems. To improve the therapeutic effect of the cultured bone, we investigated the culture conditions that contributed to extensive osteoblastic differentiation. Rat bone marrow was primarily cultured to expand the number of MSCs and further cultured in osteogenic medium for 12 days. The culture was also conducted in a medium supplemented with either bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor (FGF-2), or with sequential combinations of both supplements. Among them, the sequential supplementation of FGF-2 followed by BMP-2 showed high alkaline phosphatase activity, sufficient bone-specific osteocalcein expression and abundant bone matrix formation of the MSC culture. These data implied that the number of responding cells or immature osteoblasts was increased by the supplementation of FGF-2 in the early phase of the culture and that these cells can show osteoblastic differentiation, of which capability was augmented by BMP-2 in the late phase. The sequential supplementation of these cytokines into MSC culture might be suitable for the fabrication of ideal cultured bone for use in bone tissue engineering.

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

    PubMed Central

    Elsafadi, Mona; Manikandan, Muthurangan; Atteya, Muhammad; Hashmi, Jamil Amjad; Iqbal, Zafar; Aldahmash, Abdullah; Alfayez, Musaad

    2016-01-01

    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 heterogeneity of hBMSC cultures. In the present study, we characterized in detail two hTERT-BMSC clonal cell populations termed here CL1 and CL2 that represent an opposing phenotype with respect to morphology, markers expression: alkaline phosphatase (ALP) and CD146, and ex vivo differentiation potential. CL1 differentiated readily to osteoblasts, adipocytes, and chondrocytes as shown by expression of lineage specific genes and proteins. Whole genome transcriptome profiling of CL1 versus CL2 revealed enrichment in CL1 of bone-, mineralization-, and skeletal muscle-related genes, for example, ALP, POSTN, IGFBP5 BMP4, and CXCL12. On the other hand, CL2 transcriptome was enriched in immune modulatory genes, for example, CD14, CD99, NOTCH3, CXCL6, CFB, and CFI. Furthermore, gene expression microarray analysis of osteoblast differentiated CL1 versus CL2 showed significant upregulation in CL1 of bone development 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 and functional heterogeneity in cultured hBMSC. ALP can be employed to identify osteoblastic and adipocytic progenitor cells in the heterogeneous hBMSC cultures. PMID:27610142

  3. Human osteoblasts derived from mesenchymal stem cells express adipogenic markers upon coculture with bone marrow adipocytes.

    PubMed

    Clabaut, Aline; Delplace, Séverine; Chauveau, Christophe; Hardouin, Pierre; Broux, Odile

    2010-07-01

    In osteoporosis, bone loss is accompanied by greater adiposity in the marrow. Given the cellular proximity within the bone marrow, we wondered whether adipocytes might have a paracrine impact on osteoblast differentiation. To test this hypothesis, we cocultured adipocytes with osteoblasts derived from mesenchymal stem cells (MSCs) in the absence of direct cell contact and then analyzed gene expression changes in the osteoblastic population by using real-time reverse transcription polymerase chain reaction. We found that, upon coculture, MSC-derived osteoblasts showed appearance of adipogenic (lipoprotein lipase, leptin) and decrease of osteogenic (osteocalcin) mRNA markers. Our results indicate that in vitro, MSC-derived adipocytes are capable of inducing MSC-derived osteoblasts to differentiate to an adipocyte phenotype. These new data suggest that (i) transdifferentiation of committed osteoblasts into adipocytes may contribute to the increase in marrow fat content at the expense of bone-forming cells and (ii) this switch might be initiated by the adipocytes themselves.

  4. Supernatant of Bone Marrow Mesenchymal Stromal Cells Induces Peripheral Blood Mononuclear Cells Possessing Mesenchymal Features

    PubMed Central

    Hu, Gang; Xu, Jun-jun; Deng, Zhi-hong; Feng, Jie; Jin, Yan

    2011-01-01

    Increasing evidence shows that some cells from peripheral blood fibroblast-like mononuclear cells have the capacity to differentiate into mesenchymal lineages. However, the insufficiency of these cells in the circulation challenges the cell isolation and subsequently limits the clinical application of these cells. In the present study, the peripheral blood mononuclear cells (pbMNCs) were isolated from wound animals and treated with the supernatant of bone marrow mesenchymal stromal cells (bmMSCs). Results showed these pbMNCs were fibroblast-like, had stromal morphology, were negative for CD34 and CD45, but positive for Vimentin and Collagen I, and had the multipotency to differentiate into adipocytes and osteoblasts. We named these induced peripheral blood-derived mesenchymal stromal cells (ipbMSCs). Skin grafts in combination with ipbMSCs and collagen I were applied for wound healing, and results revealed ipbMSC exhibited similar potency and effectiveness in the promotion of wound healing to the bmMSCs. Hereafter, we speculate that the mixture of growth factors and chemokines secreted by bmMSCs may play an important roles in the induction of the proliferation and mesenchymal differentiation of mononuclear cells. Our results are clinically relevant because it provide a new method for the acquisition of MSCs which can be used as a candidate for the wound repair. PMID:21494428

  5. Metallofullerene nanoparticles promote osteogenic differentiation of bone marrow stromal cells through BMP signaling pathway

    NASA Astrophysics Data System (ADS)

    Yang, Kangning; Cao, Weipeng; Hao, Xiaohong; Xue, Xue; Zhao, Jing; Liu, Juan; Zhao, Yuliang; Meng, Jie; Sun, Baoyun; Zhang, Jinchao; Liang, Xing-Jie

    2013-01-01

    Although endohedral metallofullerenol [Gd@C82(OH)22]n nanoparticles have anti-tumor efficiency and mostly deposit in the bones of mice, how these nanoparticles act in bone marrow stromal cells (MSCs) remains largely unknown. Herein, we observed that [Gd@C82(OH)22]n nanoparticles facilitated the differentiation of MSCs toward osteoblasts, as evidenced by the enhancement of alkaline phosphatase (ALP) activity and mineralized nodule formation upon [Gd@C82(OH)22]n nanoparticle treatment. Mechanistically, the effect of [Gd@C82(OH)22]n nanoparticles on ALP activity was inhibited by the addition of noggin as an inhibitor of the BMP signaling pathway. Moreover, the in vivo results of the ovariectomized rats further indicated that [Gd@C82(OH)22]n nanoparticles effectively improved bone density and prevented osteoporosis.Although endohedral metallofullerenol [Gd@C82(OH)22]n nanoparticles have anti-tumor efficiency and mostly deposit in the bones of mice, how these nanoparticles act in bone marrow stromal cells (MSCs) remains largely unknown. Herein, we observed that [Gd@C82(OH)22]n nanoparticles facilitated the differentiation of MSCs toward osteoblasts, as evidenced by the enhancement of alkaline phosphatase (ALP) activity and mineralized nodule formation upon [Gd@C82(OH)22]n nanoparticle treatment. Mechanistically, the effect of [Gd@C82(OH)22]n nanoparticles on ALP activity was inhibited by the addition of noggin as an inhibitor of the BMP signaling pathway. Moreover, the in vivo results of the ovariectomized rats further indicated that [Gd@C82(OH)22]n nanoparticles effectively improved bone density and prevented osteoporosis. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr33575a

  6. The phytoestrogen genistein enhances osteogenesis and represses adipogenic differentiation of human primary bone marrow stromal cells.

    PubMed

    Heim, M; Frank, O; Kampmann, G; Sochocky, N; Pennimpede, T; Fuchs, P; Hunziker, W; Weber, P; Martin, I; Bendik, I

    2004-02-01

    In the present study, we investigated the role of the phytoestrogen genistein and 17beta-estradiol in human bone marrow stromal cells, undergoing induced osteogenic or adipogenic differentiation. Profiling of estrogen receptors (ERs)-alpha, -beta1, -beta2, -beta3, -beta4, -beta5, and aromatase mRNAs revealed lineage-dependent expression patterns. During osteogenic differentiation, the osteoblast-determining core binding factor-alpha1 showed a progressive increase, whereas the adipogenic regulator peroxisome proliferator-activated receptor gamma (PPARgamma) was sequentially decreased. This temporal regulation of lineage-determining marker genes was strongly enhanced by genistein during the early osteogenic phase. Moreover, genistein increased alkaline phosphatase mRNA levels and activity, the osteoprotegerin:receptor activator of nuclear factor-kappaB ligand gene expression ratio, and the expression of TGFbeta1. During adipogenic differentiation, down-regulation in the mRNA levels of PPARgamma and CCAAT/enhancer-binding protein-alpha at d 3 and decreased lipoprotein lipase and adipsin mRNA levels at d 21 were observed after genistein treatment. This led to a lower number of adipocytes and a reduction in the size of their lipid droplets. At d 3 of adipogenesis, TGFbeta1 was strongly up-regulated by genistein in an ER-dependent manner. Blocking the TGFbeta1 pathway abolished the effects of genistein on PPARgamma protein levels and led to a reduction in the proliferation rate of precursor cells. Overall, genistein enhanced the commitment and differentiation of bone marrow stromal cells to the osteoblast lineage but did not influence the late osteogenic maturation markers. Adipogenic differentiation and maturation, on the other hand, were reduced by genistein (and 17beta-estradiol) via an ER-dependent mechanism involving autocrine or paracrine TGFbeta1 signaling.

  7. Substance P stimulates bone marrow stromal cell osteogenic activity, osteoclast differentiation, and resorption activity in vitro

    PubMed Central

    Wang, Liping; Zhao, Rong; Shi, Xiaoyou; Wei, Tzuping; Halloran, Bernard P.; Clark, David J.; Jacobs, Christopher R.; Kingery, Wade S.

    2009-01-01

    Introduction SP is a neuropeptide distributed in the sensory nerve fibers that innervate the medullar tissues of bone, as well as the periosteum. Previously we demonstrated that inhibition of neuropeptide signaling after capsaicin treatment resulted in a loss of bone mass and we hypothesized that SP contributes to bone integrity by stimulating osteogenesis. Materials and Methods Osteoblast precursors (bone marrow stromal cells, BMSCs) and osteoclast precursors (bone marrow macrophages, BMMs) derived from C57BL/6 mice were cultured. Expression of the SP receptor (NK1) was detected by using immunocytochemical staining and PCR. Effects of SP on proliferation and differentiation of BMSCs were studied by measuring BrdU incorporation, gene expression, alkaline phosphatase activity, and osteocalcin and Runx2 protein levels with EIA and western blot assays, respectively. Effects of SP on BMMs were determined using a BrdU assay, counting multinucleated cells staining positive for tartrate-resistant acid phosphatase (TRAP+), measuring pit erosion area, and evaluating RANKL protein production and NF-κB activity with ELISA and western blot. Results The NK1 receptor was expressed in both BMSCs and BMMs. SP stimulated the proliferation of BMSCs in a concentration-dependent manner. Low concentrations (10−12 M) of SP stimulated alkaline phosphatase and osteocalcin expression, increased alkaline phosphatase activity, and up-regulated Runx2 protein levels, and higher concentrations of SP (10−8 M) enhanced mineralization in differentiated BMSCs. SP also stimulated BMSCs to produce RANKL, but at concentrations too low to evoke osteoclastogenesis in co-culture with macrophages in the presence of SP. SP also activated NF-κB in BMMs and directly facilitate RANKL induced macrophage osteoclastogenesis and bone resorption activity. Conclusions NK1 receptors are expressed by osteoblast and osteoclast precursors and SP stimulates osteoblast and osteoclast differentiation and function in

  8. Bone marrow stromal stem cells: nature, biology, and potential applications.

    PubMed

    Bianco, P; Riminucci, M; Gronthos, S; Robey, P G

    2001-01-01

    Bone marrow stromal cells are progenitors of skeletal tissue components such as bone, cartilage, the hematopoiesis-supporting stroma, and adipocytes. In addition, they may be experimentally induced to undergo unorthodox differentiation, possibly forming neural and myogenic cells. As such, they represent an important paradigm of post-natal nonhematopoietic stem cells, and an easy source for potential therapeutic use. Along with an overview of the basics of their biology, we discuss here their potential nature as components of the vascular wall, and the prospects for their use in local and systemic transplantation and gene therapy.

  9. Contribution of SATB2 to the stronger osteogenic potential of bone marrow stromal cells from craniofacial bones.

    PubMed

    Zhang, Ping; Men, Jie; Fu, Yu; Shan, Tengfei; Ye, Jinhai; Wu, Yunong; Tao, Zhenjiang; Liu, Laikui; Jiang, Hongbing

    2012-12-01

    Previous studies have shown that craniofacial bone marrow stromal cells (BMSCs) have a strong osteogenic potential. However, the mechanism by which BMSCs of various embryonic origins develop diverse osteogenic potentials remains unclear. To investigate the mechanisms regulating osteoblast differentiation in two different types of BMSCs, we compared the temporal and spatial mRNA and protein expression patterns of Satb2 and its downstream gene Hoxa2 by using real-time polymerase chain reaction, Western blotting and fluorescent immunostaining in mandible BMSCs (M-BMSCs) and tibia BMSCs (T-BMSCs) undergoing osteoblast differentiation. Higher levels of alkaline phosphatase, greater calcium accumulation and earlier expression of Runx2 were observed in osteogenic-induced M-BMSCs compared with T-BMSCs. Low levels of Satb2 were detected in both types of uninduced BMSCs but the majority of SATB2 was located in the nuclei of M-BMSCs. Notably, Satb2 was expressed earlier in M-BMSCs and Hoxa2, a downstream target of Satb2, was not expressed in uninduced M-BMSCs or during osteoblast differentiation, just as during embryonic mandible development. In contrast, Hoxa2 was reactivated in T-BMSCs during osteoblast differentiation. Based on these results, we conclude that SATB2 plays a different role during osteoblast differentiation of M-BMSCs and T-BMSCs. The earlier activation of Satb2 expression in M-BMSCs compared with T-BMSCs might explain the stronger osteogenic potential of M-BMSCs.

  10. Impaired Function of Bone Marrow Stromal Cells in Systemic Mastocytosis

    PubMed Central

    Nemeth, K.; Wilson, T.M.; Ren, J.J.; Sabatino, M.; Stroncek, D.F.; Krepuska, M.; Bai, Y.; Robey, P.G.; Metcalfe, D.D.; Mezey, E.

    2015-01-01

    Patients with systemic mastocytosis (SM) have a wide variety of problems, including skeletal abnormalities. The disease results from a mutation of the stem cell receptor (c-kit) in mast cells and we wondered if the function of bone marrow stromal cells (BMSCs; also known as MSCs or mesenchymal stem cells) might be affected by the invasion of bone marrow by mutant mast cells. As expected, BMSCs from SM patients do not have a mutation in c-kit, but they proliferate poorly. In addition, while osteogenic differentiation of the BMSCs seems to be deficient, their adipogenic potential appears to be increased. Since the hematopoietic supportive abilities of BMSCs are also important, we also studied the engraftment in NSG mice of human CD34+ hematopoietic progenitors, after being co-cultured with BMSCs of healthy volunteers vs. BMSCs derived from patients with SM. BMSCs derived from the bone marrow of patients with SM could not support hematopoiesis to the extent that healthy BMSCs do. Finally, we performed an expression analysis and found significant differences between healthy and SM derived BMSCs in the expression of genes with a variety of functions, including the WNT signaling, ossification, and bone remodeling. We suggest that some of the symptoms associated with SM might be driven by epigenetic changes in BMSCs caused by dysfunctional mast cells in the bone marrow of the patients. PMID:26001169

  11. Engraftment Outcomes after HPC Co-Culture with Mesenchymal Stromal Cells and Osteoblasts.

    PubMed

    Cook, Matthew M; Doran, Michael R; Kollar, Katarina; Barbier, Valerie; Winkler, Ingrid G; Levesque, Jean-Pierre; Brooke, Gary; Atkinson, Kerry

    2013-09-23

    Haematopoietic stem cell (HSC) transplantation is an established cell-based therapy for a number of haematological diseases. To enhance this therapy, there is considerable interest in expanding HSCs in artificial niches prior to transplantation. This study compared murine HSC expansion supported through co-culture on monolayers of either undifferentiated mesenchymal stromal cells (MSCs) or osteoblasts. Sorted Lineage(-) Sca-1(+) c-kit(+) (LSK) haematopoietic stem/progenitor cells (HPC) demonstrated proliferative capacity on both stromal monolayers with the greatest expansion of LSK shown in cultures supported by osteoblast monolayers. After transplantation, both types of bulk-expanded cultures were capable of engrafting and repopulating lethally irradiated primary and secondary murine recipients. LSKs co-cultured on MSCs showed comparable, but not superior, reconstitution ability to that of freshly isolated LSKs. Surprisingly, however, osteoblast co-cultured LSKs showed significantly poorer haematopoietic reconstitution compared to LSKs co-cultured on MSCs, likely due to a delay in short-term reconstitution. We demonstrated that stromal monolayers can be used to maintain, but not expand, functional HSCs without a need for additional haematopoietic growth factors. We also demonstrated that despite apparently superior in vitro performance, co-injection of bulk cultures of osteoblasts and LSKs in vivo was detrimental to recipient survival and should be avoided in translation to clinical practice.

  12. Characterization of bone marrow mesenchymal stromal cells in aplastic anaemia.

    PubMed

    Hamzic, Edita; Whiting, Karen; Gordon Smith, Edward; Pettengell, Ruth

    2015-06-01

    In aplastic anaemia (AA), haemopoietic activity is significantly reduced and generally attributed to failure of haemopoietic stem cells (HSC) within the bone marrow (BM). The regulation of haemopoiesis depends on the interaction between HSC and various cells of the BM microenvironment, including mesenchymal stromal cells (MSC). MSC involvement in the functional restriction of HSC in AA is largely unknown and therefore, the physical and functional properties of AA MSC were studied in vitro. MSC were characterized by their phenotype and ability to form adherent stromal layers. The functional properties of AA MSC were assessed through proliferative, clonogenic and cross-over culture assays. Results indicate that although AA MSC presented typical morphology and distinctive mesenchymal markers, stromal formation was reduced, with 50% of BM samples failing to produce adherent layers. Furthermore, their proliferative and clonogenic capacity was markedly decreased (P = 0·03 and P = 0·04 respectively) and the ability to sustain haemopoiesis was significantly reduced, as assessed by total cell proliferation (P = 0·032 and P = 0·019 at Week 5 and 6, respectively) and clonogenic potential of HSC (P = 0·02 at Week 6). It was concluded that the biological characteristics of AA MSC are different from those of control MSC and their in vitro haemopoiesis-supporting ability is significantly reduced.

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

    SciTech Connect

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

    2009-03-20

    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.

  14. Pleiotrophin Commits Human Bone Marrow Mesenchymal Stromal Cells towards Hypertrophy during Chondrogenesis

    PubMed Central

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

    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. PMID:24516627

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

    PubMed

    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.

  16. Differential expression of collagenase by human fibroblasts and bone marrow stromal cells.

    PubMed

    Takahashi, G W; Moran, D; Andrews, D F; Singer, J W

    1994-02-01

    The bone marrow stroma, represented in long-term marrow culture by cells of the adherent layer, is composed of a heterogenous mixture of macrophages and mesenchymal cells, including fibroblasts, endothelial cells and adipocytes, in association with a proteoglycan matrix. This matrix, which is synthesized by the stroma, is capable of binding hematopoietic growth factors, and likely plays a major role in hematopoietic regulation. Clonally-derived non-transformed bone marrow stromal cells, propagated in the presence of basic fibroblast growth factor, were studied for expression of collagenase, an enzyme whose substrate, collagen, is a major component of the extracellular matrix. Expression of steady-state collagenase mRNA was undetectable in both unstimulated dermal fibroblasts and non-transformed marrow stromal cells. However, stimulation with interleukin 1 alpha (10 U/ml) for 24 h resulted in marked accumulation of collagenase mRNA in dermal fibroblast cells, yet failed to elicit a similar response in bone marrow stromal cells. Both marrow stromal cells and dermal fibroblasts constitutively expressed transcripts of collagen I, and rhIL-1 alpha upregulated transcripts of interleukin 6 in both these cells as well. Although similar in morphology, these data indicate that bone marrow stromal cells differ from fibroblasts in their response to IL-1. In the marrow microenvironment, where IL-1 may be secreted by a variety of cell types, such suppression of collagenase expression may serve to prevent unwanted mobilization of collagen from the glycoprotein matrix by marrow stromal cells.

  17. Age-related BMAL1 change affects mouse bone marrow stromal cell proliferation and osteo-differentiation potential

    PubMed Central

    Chen, Yijia; Xu, Xiaomei; Tan, Zhen; Ye, Cui; Chen, Yangxi

    2012-01-01

    Introduction Aging people's bone regeneration potential is always impaired. Bone marrow stromal cells (MSCs) contain progenitors of osteoblasts. Donor age may affect MSCs’ proliferation and differentiation potential, but the genomic base is still unknown. Due to recent research's indication that a core circadian component, brain and muscle ARNT-like 1 protein (BMAL1), has a role in premature aging, we investigated the normal aging mechanism in mice with their MSCs and Bmal1 gene/protein level. Material and methods 1, 6 and 16 month old C57BL/6 mice were used and the bone marrow stromal cells were gained and cultured at early passage. Bmal1 gene and protein level were detected in these cells. Marrow stromal cells were also induced to differentiate to osteoblasts or adipocytes. Three groups of mice MSCs were compared on proliferation by flow cytometry, on cell senescence by SA-β-gal expression and after osteo-induction on osteogenic potential by the expression of osterix (Osx), alkaline phosphatase (ALP) and osteocalcin (OCN). Results Bmal1 gene and protein level as well as S-phase fraction of the cell cycle decreased in MSCs along with the aging process. At the same time, SA-β-gal+ levels increased, especially in the aged mice MSCs. When induced to be osteogenic, Osx gene expression and ALP activity declined in the mid-age and aged mice MSCs, while OCN protein secretion deteriorated in the aged mice MSCs. Conclusions These findings demonstrate that mouse MSCs changed with their proliferation and osteo-differentiation abilities at different aging stages, and that Bmal1 is related to the normal aging process in MSCs. PMID:22457671

  18. Adipose-derived mesenchymal stromal (stem) cells differentiate to osteoblast and chondroblast lineages upon incubation with conditioned media from dental pulp stem cell-derived osteoblasts and auricle cartilage chondrocytes.

    PubMed

    Carbone, A; Valente, M; Annacontini, L; Castellani, S; Di Gioia, S; Parisi, D; Rucci, M; Belgiovine, G; Colombo, C; Di Benedetto, A; Mori, G; Lo Muzio, L; Maiorella, A; Portincasa, A; Conese, M

    2016-01-01

    The potential of adipose-derived mesenchymal stromal (stem) cells (ADSCs) to differentiate into either osteoblasts or chondrocytes is controversial. In this study we investigated the multicapacity potential of ADSCs to differentiate towards adipocyte, osteoblast, and chondrocyte lineages when cells are seeded onto plastic in comparison with incubation with conditioned media (CM) obtained from differentiated cell types.ADSCs, obtained from liposuctions, were characterized for mesenchymal and hematopoietic markers by cytofluorimetry. Their differentiation capacity towards adipocytes, osteoblasts, and chondrocytes was investigated by histochemistry methods (Oil-Red-O staining, Safranin O and Alizarin Red staining, respectively). Dental pulp stem cells (DPSCs) and dedifferentiated auricle derived-chondrocytes were differentiated towards osteoblastic and chondrocytic lineages respectively, and the CM obtained from these cultures was used to induce differentiation of ADSCs. ADSCs were positive for mesenchymal markers (CD29, CD105, CD73, CD44), but not for hematopoietic lineage markers (CD14, CD34, CD45) and this behavior was conserved from the isolation up to the fifth passage. While ADSCs were readily differentiated in adipocytes, they were not towards chondrocytes and osteoblastic lineages, a behavior different from that of bone marrow-derived MSCs that differentiated into the three lineages at two weeks post-induction. Only ADSCs treated with CM from cultured chondrocytes and DPSCs, produced glycosaminoglycans and mineralized matrix. These results indicate that ADSCs need growth/morphogenic factor supplementation from the tissue environment to be appropriately differentiated to mesodermic lineages.

  19. Stromal cell-derived factor-1 mediates changes of bone marrow stem cells during the bone repair process.

    PubMed

    Okada, Kiyotaka; Kawao, Naoyuki; Yano, Masato; Tamura, Yukinori; Kurashimo, Shinzi; Okumoto, Katsumi; Kojima, Kotarou; Kaji, Hiroshi

    2016-01-01

    Osteoblasts, osteoclasts, chondrocytes, and macrophages that participate in the bone repair process are derived from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the roles of these stem cells during the repair of injured bone tissue are still unclear. In the present study, we examined the effects of bone defect on HSCs and MSCs in bone marrow and spleen in 75 mice and its mechanism. We analyzed the HSC and MSC populations in these tissues of a mouse with femoral bone damage by using flow cytometry. The number of HSCs in the bone marrow of mice with damaged femurs was significantly lower than the number of these cells in the bone marrow of the contralateral intact femurs on day 2 after injury. Meanwhile, the number of MSCs in the bone marrow of mice with damaged femurs was significantly higher than that of the contralateral femurs. Both intraperitoneal administration of AMD3100, a C-X-C chemokine receptor 4 (CXCR4) antagonist, and local treatment with an anti-stromal cell-derived factor-1 (SDF-1) antibody blunted the observed decrease in HSC and increase in MSC populations within the bone marrow of injured femurs. In conclusion, the present study revealed that there is a concurrent decrease and increase in the numbers of HSCs and MSCs, respectively, in the bone marrow during repair of mouse femoral bone damage. Furthermore, the SDF-1/CXCR4 system was implicated as contributing to the changes in these stem cell populations upon bone injury.

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

    PubMed

    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.

  1. Mouse embryonic fibroblasts (MEF) exhibit a similar but not identical phenotype to bone marrow stromal stem cells (BMSC).

    PubMed

    Saeed, Hamid; Taipaleenmäki, Hanna; Aldahmash, Abdullah M; Abdallah, Basem M; Kassem, Moustapha

    2012-06-01

    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 reported. Utilizing standard in vitro and in vivo assays we performed a side-by-side comparison of MEF and BMSC to determine their ability to differentiate into mesoderm-type cells. BMSC were isolated from 8-10 weeks old mouse bone marrow by plastic adherence. MEF were established by trypsin/EDTA digestion from E13.5 embryos after removing heads and viscera, followed by plastic adherence. Compared to BMSC, MEF exhibited telomerase activity and improved cell proliferation as assessed by q-PCR based TRAP assay and cell number quantification, respectively. FACS analysis revealed that MEF exhibited surface markers characteristic of the BMSC: Sca-1(+), CD73(+), CD105(+), CD29(+), CD44(+), CD106(+), CD11b(-), and CD45(-). In contrast to BMSC, ex vivo osteoblast (OB) differentiation of MEF exhibited a less mature osteoblastic phenotype (less alkaline phosphatase, collagen type I and osteocalcin) as assessed 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/tricalcium phosphate, in immune deficient mice. In conclusion, MEF contain a population of stem cells that behave in ex vivo and in vivo assays, similar but not identical, to BMSC. Due to their enhanced cell growth, they may represent a good alternative for BMSC in studying molecular mechanisms of stem cell commitment and differentiation to osteoblasts, adipocytes and chondrocytes.

  2. Cardiomyocytes can be generated from marrow stromal cells in vitro

    PubMed Central

    Makino, Shinji; Fukuda, Keiichi; Miyoshi, Shunichirou; Konishi, Fusako; Kodama, Hiroaki; Pan, Jing; Sano, Motoaki; Takahashi, Toshiyuki; Hori, Shingo; Abe, Hitoshi; Hata, Jun-ichi; Umezawa, Akihiro; Ogawa, Satoshi

    1999-01-01

    We have isolated a cardiomyogenic cell line (CMG) from murine bone marrow stromal cells. Stromal cells were immortalized, treated with 5-azacytidine, and spontaneously beating cells were repeatedly screened. The cells showed a fibroblast-like morphology, but the morphology changed after 5-azacytidine treatment in ∼30% of the cells; they connected with adjoining cells after one week, formed myotube-like structures, began spontaneously beating after two weeks, and beat synchronously after three weeks. They expressed atrial natriuretic peptide and brain natriuretic peptide and were stained with anti-myosin, anti-desmin, and anti-actinin antibodies. Electron microscopy revealed a cardiomyocyte-like ultrastructure, including typical sarcomeres, a centrally positioned nucleus, and atrial granules. These cells had several types of action potentials, such as sinus node–like and ventricular cell–like action potentials. All cells had a long action potential duration or plateau, a relatively shallow resting membrane potential, and a pacemaker-like late diastolic slow depolarization. Analysis of the isoform of contractile protein genes, such as myosin heavy chain, myosin light chain, and α-actin, indicated that their muscle phenotype was similar to that of fetal ventricular cardiomyocytes. These cells expressed Nkx2.5/Csx, GATA4, TEF-1, and MEF-2C mRNA before 5-azacytidine treatment and expressed MEF-2A and MEF-2D after treatment. This new cell line provides a powerful model for the study of cardiomyocyte differentiation. J. Clin. Invest. 103:697–705 (1999) PMID:10074487

  3. Canine Cranial Reconstruction Using Autologous Bone Marrow Stromal Cells

    PubMed Central

    Mankani, Mahesh H.; Kuznetsov, Sergei A.; Shannon, Brian; Nalla, Ravi K.; Ritchie, Robert O.; Qin, Yixian; Robey, Pamela Gehron

    2006-01-01

    Limited-sized transplants of culture-expanded autologous or allogeneic bone marrow stromal cells (BMSCs) form cortico-cancellous bone in rodent models. Initiation of clinical studies using autologous BMSC transplantation requires effective bone formation among sizable transplants in a large animal model as well as noninvasive techniques for evaluating transplant success. Here, we obtained bone marrow from the femurs of six dogs and expanded BMSCs in tissue culture. Autologous BMSC-hydroxyapatite/tricalcium phosphate (HA/TCP) transplants were introduced into critical-sized calvarial defects and contralateral control skull defects received HA/TCP vehicle alone. At intervals ranging from 2 to 20 months, transplants were biopsied or harvested for histological and mechanical analysis. Noninvasive studies, including quantitative computed tomography scans and ultrasound, were simultaneously obtained. In all animals, BMSC-containing transplants formed significantly more bone than their control counterparts. BMSC-associated bone possessed mechanical properties similar to the adjacent normal bone, confirmed by both ultrasound and ex vivo analysis. Evaluation by quantitative computed tomography confirmed that the extent of bone formation demonstrated by histology could be discerned through noninvasive means. These results show that autologous cultured BMSC transplantation is a feasible therapy in clinical-sized bone defects and that such transplants can be assessed noninvasively, suggesting that this technique has potential for use in patients with certain bone defects. PMID:16436668

  4. 3D-Cultivation of bone marrow stromal cells on hydroxyapatite scaffolds fabricated by dispense-plotting and negative mould technique.

    PubMed

    Detsch, R; Uhl, F; Deisinger, U; Ziegler, G

    2008-04-01

    The main principle of a bone tissue engineering (BTE) strategy is to cultivate osteogenic cells in an osteoconductive porous scaffold. Ceramic implants for osteogenesis are based mainly on hydroxyapatite (HA), since this is the inorganic component of bone. Rapid Prototyping (RP) is a new technology in research for producing ceramic scaffolds. This technology is particularly suitable for the fabrication of individually and specially tailored single implants. For tissue engineering these scaffolds are seeded with osteoblast or osteoblast precursor cells. To supply the cultured osteoblastic cells efficiently with nutrition in these 3D-geometries a bioreactor system can be used. The aim of this study was to analyse the influence of differently fabricated HA-scaffolds on bone marrow stromal cells. For this, two RP-techniques, dispense-plotting and a negative mould method, were used to produce porous ceramics. The manufactured HA-scaffolds were then cultivated in a dynamic system (bioreactor) with an osteoblastic precursor cell line. In our study, the applied RP-techniques give the opportunity to design and process HA-scaffolds with defined porosity, interconnectivity and 3D pore distribution. A higher differentiation of bone marrow stromal cells could be detected on the negative mould fabricated scaffolds, while cell proliferation was higher on the dispense-plotted scaffolds. Nevertheless, both scaffold types can be used in tissue engineering applications.

  5. The use of catalytic carbon deposits as 3D carriers for human bone marrow stromal cells.

    PubMed

    Petrenko, Yu A; Gurin, I V; Volkova, N A; Mazur, S P; Sandomirskii, B P

    2011-08-01

    We studied the possibility of using 3D structures based on carbon catalytic deposits as carriers for human bone marrow stromal cells. It was found that carbon catalytic deposits obtained by gas deposition method using FeCl(3) × 6H(2)O as the catalyst are a biocompatible material for human bone marrow stromal cells promoting adhesion, proliferation, and distribution of cells within the 3D carrier, and therefore can be used for tissue engineering.

  6. Effects of Ti, PMMA, UHMWPE, and Co-Cr wear particles on differentiation and functions of bone marrow stromal cells

    PubMed Central

    Jiang, Yunpeng; Jia, Tanghong; Gong, Weiming; Wooley, Paul H.; Yang, Shang-You

    2013-01-01

    The current study investigates the roles of orthopaedic biomaterial particles (Ti, PMMA, UHMWPE, Co-Cr) on the differentiation and functions of bone marrow stromal cells (BMSCs). Cells were isolated from femurs of BALB/c mice and cultured in complete osteoblast-induction medium in presence of micron-sized biomaterial particles at various doses. MTT assay and lactate dehydrogenase (LDH) assay were performed for cell proliferation and cytotoxicity. Differentiation and function of osteoblasts were evaluated by alkaline phosphatase (ALP), osteocalcin, RANKL, OSX, and Runx2 expressions. Murine IL-1, IL-6 and TNFα in culture media were determined by ELISA. Challenge with low doses of Ti, UHMWPE, or Co-Cr particles markedly promoted the bone marrow cell proliferation while high dose of Co-Cr significantly inhibited cell growth (p<0.05). Cells challenged with low dose of PMMA or UHMWPE particles (0.63 mg/ml) exhibited strong ALP activity, whereas Ti, and Co-Cr groups showed minimal effects (p<0.05). UHMWPE and Ti particles also promoted higher expression of pro-inflammatory cytokines. Real-time PCR data suggested that cells treated with low dose (0.5mg/ml) particles resulted in distinctly diminished RANKL expression compared to those exposed to high concentrated (3mg/ml) particles. In conclusion, various types of wear debris particles behaved differently in the differentiation, maturation, and functions of osteogenic cells; and the particulate debris-interacted bone marrow stromal cells may play an important role in the pathogenesis and process of the debris-associated aseptic prosthetic loosening. PMID:24039045

  7. Adhesion and migration of marrow-derived osteoblasts on injectable in situ crosslinkable poly(propylene fumarate-co-ethylene glycol)-based hydrogels with a covalently linked RGDS peptide.

    PubMed

    Behravesh, Esfandiar; Zygourakis, Kyriacos; Mikos, Antonios G

    2003-05-01

    Marrow-derived osteoblasts were cultured on poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)) based hydrogels modified in bulk with a covalently linked RGDS model peptide. A poly(ethylene glycol) spacer arm was utilized to covalently link the peptide to the hydrogel. Three P(PF-co-EG) block copolymers were synthesized with varying poly(ethylene glycol) block lengths relative to poly(ethylene glycol) spacer arm. A poly(ethylene glycol) block length of nominal molecular weight 2000 and spacer arm of nominal molecular weight 3400 were found to reduce nonspecific cell adhesion and show RGDS concentration dependent marrow-derived osteoblast adhesion. A concentration of 100 nmol/mL RGDS was sufficient to promote adhesion of 84 +/- 17% of the initial seeded marrow-derived osteoblasts compared with 9 +/- 1% for the unmodified hydrogel after 12 h. Cell spreading was quantified as a method for evaluating adhesivity of cells to the hydrogel. A megacolony migration assay was utilized to assess the migration characteristics of the marrow-derived osteoblasts on RGDS modified hydrogels. Marrow-stromal osteoblasts migration was greater on hydrogels modified with 100 nmol/mL linked RGDS when compared with hydrogels modified with 1000 nmol/mL linked RGDS, while proliferation was not affected. These P(PF-co-EG) hydrogels modified in the bulk with RGDS peptide are potential candidates as in situ forming scaffolds for bone tissue engineering applications.

  8. Leukemia cells induce changes in human bone marrow stromal cells

    PubMed Central

    2013-01-01

    Background Bone marrow stromal cells (BMSCs) are multipotent cells that support angiogenesis, wound healing, and immunomodulation. In the hematopoietic niche, they nurture hematopoietic cells, leukemia, tumors and metastasis. BMSCs secrete of a wide range of cytokines, growth factors and matrix proteins which contribute to the pro-tumorigenic marrow microenvironment. The inflammatory cytokines IFN-γ and TNF-α change the BMSC secretome and we hypothesized that factors produced by tumors or leukemia would also affect the BMSC secretome and investigated the interaction of leukemia cells with BMSCs. Methods BMSCs from healthy subjects were co-cultured with three myeloid leukemia cell lines (TF-1, TF-1α and K562) using a trans-well system. Following co-culture, the BMSCs and leukemia cells were analyzed by global gene expression analysis and culture supernatants were analyzed for protein expression. As a control, CD34+ cells were also cocultured with BMSCs. Results Co-culture induced leukemia cell gene expression changes in stem cell pluripotency, TGF-β signaling and carcinoma signaling pathways. BMSCs co-cultured with leukemia cells up-regulated a number of proinflammatory genes including IL-17 signaling-related genes and IL-8 and CCL2 levels were increased in co-culture supernatants. In contrast, purine metabolism, mTOR signaling and EIF2 signaling pathways genes were up-regulated in BMSCs co-cultured with CD34+ cells. Conclusions BMSCs react to the presence of leukemia cells undergoing changes in the cytokine and chemokine secretion profiles. Thus, BMSCs and leukemia cells both contribute to the creation of a competitive niche more favorable for leukemia stem cells. PMID:24304929

  9. Phenotypic and Functional Characterization of Human Bone Marrow Stromal Cells in Hollow Fiber Bioreactors

    PubMed Central

    Li, Matthew; Tilles, Arno W.; Milwid, Jack M.; Hammad, Mohamed; Lee, Jungwoo; Yarmush, Martin L.; Parekkadan, Biju

    2011-01-01

    The transplantation of human bone marrow stromal cells (BMSCs) is a novel immunotherapeutic approach that is currently being explored in many clinical settings. Evidence suggests that the efficacy of cell transplantation is directly associated with soluble factors released by human BMSCs. In order to harness these secreted factors, we integrated BMSCs into large-scale hollow-fiber bioreactor devices in which the cells (separated by a semipermeable polyethersulfone (PES) membrane) can directly and continuously release therapeutic factors into the blood stream. BMSCs were found to be rapidly adherent and exhibited long-term viability on PES fibers. The cells also preserved their immunophenotype under physiologic fluid flow rates in the bioreactor, and exhibited no signs of differentiation during device operation, but still retained the capacity to differentiate into osteoblastic lineages. BMSC devices released growth factors and cytokines at comparable levels on a per cell basis to conventional cell culture platforms. Finally, we utilized a potency assay to demonstrate the therapeutic potential of the collected secreted factors from the BMSC devices. In summary, we have shown that culturing BMSCs in a large-scale hollow fiber bioreactor is feasible without deleterious effects on phenotype, thus providing a platform for collecting and delivering the paracrine secretions of these cells. PMID:21710576

  10. Markers for Characterization of Bone Marrow Multipotential Stromal Cells

    PubMed Central

    Boxall, Sally A.; Jones, Elena

    2012-01-01

    Given the observed efficacy of culture-expanded multipotential stromal cells, also termed mesenchymal stem cells (MSCs), in the treatment of graft-versus host and cardiac disease, it remains surprising that purity and potency characterization of manufactured cell batches remains rather basic. In this paper, we will initially discuss surface and molecular markers that were proposed to serve as the indicators of the MSC potency, in terms of their proliferative potential or the ability to differentiate into desired lineages. The second part of this paper will be dedicated to a critical discussion of surface markers of uncultured (i.e., native) bone marrow (BM) MSCs. Although no formal consensus has yet been reached on which markers may be best suited for prospective BM MSC isolation, markers that cross-react with MSCs of animal models (such as CD271 and W8-B2/MSCA-1) may have the strongest translational value. Whereas small animal models are needed to discover the in vivo function on these markers, large animal models are required for safety and efficacy testing of isolated MSCs, particularly in the field of bone and cartilage tissue engineering. PMID:22666272

  11. Effects of the 1, 4-dihydropyridine L-type calcium channel blocker benidipine on bone marrow stromal cells.

    PubMed

    Ma, Zhong-ping; Liao, Jia-cheng; Zhao, Chang; Cai, Dao-zhang

    2015-08-01

    Osteoporosis (OP) often increases the risk of bone fracture and other complications and is a major clinical problem. Previous studies have found that high blood pressure is associated with bone formation abnormalities, resulting in increased calcium loss. We have investigated the effect of the antihypertensive drug benidipine on bone marrow stromal cell (BMSC) differentiation into osteoblasts and bone formation under osteoporotic conditions. We used a combination of in vitro and in vivo approaches to test the hypothesis that benidipine promotes murine BMSC differentiation into osteoblasts. Alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), β-catenin, and low-density lipoprotein receptor-related protein 5 (LRP5) protein expression was evaluated in primary femoral BMSCs from C57/BL6 mice cultured under osteogenic conditions for 2 weeks to examine the effects of benidipine. An ovariectomized (OVX) mouse model was used to investigate the effect of benidipine treatment for 3 months in vivo. We found that ALP, OCN, and RUNX2 expression was up-regulated and WNT/β-catenin signaling was enhanced in vitro and in vivo. In OVX mice that were intragastrically administered benidipine, bone parameters (trabecular thickness, bone mineral density, and trabecular number) in the distal femoral metaphysis were significantly increased compared with control OVX mice. Consistently, benidipine promoted BMSC differentiation into osteoblasts and protected against bone loss in OVX mice. Therefore, benidipine might be a suitable candidate for the treatment of patients with postmenopausal osteoporosis and hypertension.

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

    SciTech Connect

    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/sub 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/sub 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.

  13. Human Bone Marrow Stromal Cells: A Reliable, Challenging Tool for In Vitro Osteogenesis and Bone Tissue Engineering Approaches

    PubMed Central

    Hempel, Ute; Müller, Katrin; Preissler, Carolin; Noack, Carolin; Boxberger, Sabine; Dieter, Peter; Bornhäuser, Martin; Wobus, Manja

    2016-01-01

    Adult human bone marrow stromal cells (hBMSC) are important for many scientific purposes because of their multipotency, availability, and relatively easy handling. They are frequently used to study osteogenesis in vitro. Most commonly, hBMSC are isolated from bone marrow aspirates collected in clinical routine and cultured under the “aspect plastic adherence” without any further selection. Owing to the random donor population, they show a broad heterogeneity. Here, the osteogenic differentiation potential of 531 hBMSC was analyzed. The data were supplied to correlation analysis involving donor age, gender, and body mass index. hBMSC preparations were characterized as follows: (a) how many passages the osteogenic characteristics are stable in and (b) the influence of supplements and culture duration on osteogenic parameters (tissue nonspecific alkaline phosphatase (TNAP), octamer binding transcription factor 4, core-binding factor alpha-1, parathyroid hormone receptor, bone gla protein, and peroxisome proliferator-activated protein γ). The results show that no strong prediction could be made from donor data to the osteogenic differentiation potential; only the ratio of induced TNAP to endogenous TNAP could be a reliable criterion. The results give evidence that hBMSC cultures are stable until passage 7 without substantial loss of differentiation potential and that established differentiation protocols lead to osteoblast-like cells but not to fully authentic osteoblasts. PMID:27293446

  14. Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

    PubMed

    Besse, A; Trimoreau, F; Faucher, J L; Praloran, V; Denizot, Y

    1999-07-08

    Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.

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

    SciTech Connect

    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

    2012-11-15

    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.

  16. Molecular Signature and In Vivo Behavior of Bone Marrow Endosteal and Subendosteal Stromal Cell Populations and their Relevance to Hematopoiesis

    PubMed Central

    Balduino, Alex; Coelho, Valeria Mello; Wang, Zhou; Taichman, Russell S.; Krebsbach, Paul H.; Weeraratna, Ashani T.; Becker, Kevin G.; de Mello, Wallace; 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. PMID:22841688

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

    PubMed Central

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

    2014-01-01

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

  18. Engineering injectable bone using bone marrow stromal cell aggregates.

    PubMed

    Ma, Dongyang; Zhong, Cuiping; Yao, Hong; Liu, Yanpu; Chen, Fulin; Li, Jianxue; Zhao, Jinlong; Mao, Tianqiu; Ren, Liling

    2011-06-01

    With the increasing popularity of minimally invasive surgery, to develop an injectable bone would be highly preferable for the repair of bone nonunions and defects. However, the use of dissociated cells and exogenous carriers to construct injectable bone faces several drawbacks. To circumvent these limitations, we first harvested a cell sheet from rabbit bone marrow stromal cells using a continuous culture method and a scraping technique. The obtained sheet was then cut into fragments of multicellular aggregates, each of which was composed of a certain number of cells, extracellular matrix, and intercellular connections. The aggregates showed apparent mineralization properties, high alkaline phosphatase activity, increased osteocalcin content, and upregulated bone markers, implying their in vitro osteogenic potential. Then, serum-free medium (the control group), dissociated cell suspension (the cell group), and suspension of multicellular aggregates (the aggregate group) were injected subcutaneously on the back of the nude mice to evaluate ectopic bone formation. The results revealed that the aggregate group showed significantly larger and denser bone at the injection sites than the cell group, whereas bone formation did not occur in the control group. Additionally, when injecting them locally into the mandibular fracture gap of delayed healing in a rabbit model, we observed the most improved bone healing in the aggregate group. More cells survive and retain at the injection sites in the aggregate group than that in the cell group postoperatively. Our study indicates that the multicellular aggregates might be considered a promising strategy to generate injectable bone tissue and improve the efficacy of cell therapy.

  19. Osteoblastic Wnts differentially regulate bone remodeling and the maintenance of bone marrow mesenchymal stem cells.

    PubMed

    Wan, Yong; Lu, Cheng; Cao, Jingjing; Zhou, Rujiang; Yao, Yiyun; Yu, Jian; Zhang, Lingling; Zhao, Haixia; Li, Hanjun; Zhao, Jianzhi; Zhu, Xuming; He, Lin; Liu, Yongzhong; Yao, Zhengju; Yang, Xiao; Guo, Xizhi

    2013-07-01

    Wnt signaling has important roles in embryonic bone development and postnatal bone remodeling, but inconsistent impact on bone property is observed in different genetic alterations of Lrp5 and β-catenin. More importantly, it is still controversial whether Lrp5 regulate bone formation locally or globally through gut-derived serotonin. Here we explored the function of Wnt proteins in osteoblastic niche through inactivation of the Wntless (Wls) gene, which abrogates the secretion of Wnts. The depletion of Wls in osteoblast progenitor cells resulted in severe osteopenia with more profound defects in osteoblastogenesis, osteoclastogenesis and maintenance of bone marrow mesenchymal stem cells (BMSCs) compared to that observed in Lrp5 and β-catenin mutants. These findings support the point of view that Wnt/Lrp5 signaling locally regulates bone mass accrual through multiple effects of osteoblastic Wnts on osteoblastic bone formation and osteoclastic bone resorption. Moreover, osteoblastic Wnts confer a niche role for maintenance of BMSCs, providing novel cues for the definition of BMSCs niche in bone marrow.

  20. Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds

    NASA Technical Reports Server (NTRS)

    Ishaug, S. L.; Crane, G. M.; Miller, M. J.; Yasko, A. W.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    1997-01-01

    Bone formation was investigated in vitro by culturing stromal osteoblasts in three-dimensional (3-D), biodegradable poly(DL-lactic-co-glycolic acid) foams. Three polymer foam pore sizes, ranging from 150-300, 300-500, and 500-710 microns, and two different cell seeding densities, 6.83 x 10(5) cells/cm2 and 22.1 x 10(5) cells/cm2, were examined over a 56-day culture period. The polymer foams supported the proliferation of seeded osteoblasts as well as their differentiated function, as demonstrated by high alkaline phosphatase activity and deposition of a mineralized matrix by the cells. Cell number, alkaline phosphatase activity, and mineral deposition increased significantly over time for all the polymer foams. Osteoblast foam constructs created by seeding 6.83 x 10(5) cells/cm2 on foams with 300-500 microns pores resulted in a cell density of 4.63 x 10(5) cells/cm2 after 1 day in culture; they had alkaline phosphatase activities of 4.28 x 10(-7) and 2.91 x 10(-6) mumol/cell/min on Days 7 and 28, respectively; and they had a cell density that increased to 18.7 x 10(5) cells/cm2 by Day 56. For the same constructs, the mineralized matrix reached a maximum penetration depth of 240 microns from the top surface of the foam and a value of 0.083 mm for mineralized tissue volume per unit of cross sectional area. Seeding density was an important parameter for the constructs, but pore size over the range tested did not affect cell proliferation or function. This study suggests the feasibility of using poly(alpha-hydroxy ester) foams as scaffolding materials for the transplantation of autogenous osteoblasts to regenerate bone tissue.

  1. Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds.

    PubMed

    Ishaug, S L; Crane, G M; Miller, M J; Yasko, A W; Yaszemski, M J; Mikos, A G

    1997-07-01

    Bone formation was investigated in vitro by culturing stromal osteoblasts in three-dimensional (3-D), biodegradable poly(DL-lactic-co-glycolic acid) foams. Three polymer foam pore sizes, ranging from 150-300, 300-500, and 500-710 microns, and two different cell seeding densities, 6.83 x 10(5) cells/cm2 and 22.1 x 10(5) cells/cm2, were examined over a 56-day culture period. The polymer foams supported the proliferation of seeded osteoblasts as well as their differentiated function, as demonstrated by high alkaline phosphatase activity and deposition of a mineralized matrix by the cells. Cell number, alkaline phosphatase activity, and mineral deposition increased significantly over time for all the polymer foams. Osteoblast foam constructs created by seeding 6.83 x 10(5) cells/cm2 on foams with 300-500 microns pores resulted in a cell density of 4.63 x 10(5) cells/cm2 after 1 day in culture; they had alkaline phosphatase activities of 4.28 x 10(-7) and 2.91 x 10(-6) mumol/cell/min on Days 7 and 28, respectively; and they had a cell density that increased to 18.7 x 10(5) cells/cm2 by Day 56. For the same constructs, the mineralized matrix reached a maximum penetration depth of 240 microns from the top surface of the foam and a value of 0.083 mm for mineralized tissue volume per unit of cross sectional area. Seeding density was an important parameter for the constructs, but pore size over the range tested did not affect cell proliferation or function. This study suggests the feasibility of using poly(alpha-hydroxy ester) foams as scaffolding materials for the transplantation of autogenous osteoblasts to regenerate bone tissue.

  2. Bone formation by three-dimensional stromal osteoblast culture in biodegradable polymer scaffolds

    NASA Technical Reports Server (NTRS)

    Ishaug, S. L.; Crane, G. M.; Miller, M. J.; Yasko, A. W.; Yaszemski, M. J.; Mikos, A. G.; McIntire, L. V. (Principal Investigator)

    1997-01-01

    Bone formation was investigated in vitro by culturing stromal osteoblasts in three-dimensional (3-D), biodegradable poly(DL-lactic-co-glycolic acid) foams. Three polymer foam pore sizes, ranging from 150-300, 300-500, and 500-710 microns, and two different cell seeding densities, 6.83 x 10(5) cells/cm2 and 22.1 x 10(5) cells/cm2, were examined over a 56-day culture period. The polymer foams supported the proliferation of seeded osteoblasts as well as their differentiated function, as demonstrated by high alkaline phosphatase activity and deposition of a mineralized matrix by the cells. Cell number, alkaline phosphatase activity, and mineral deposition increased significantly over time for all the polymer foams. Osteoblast foam constructs created by seeding 6.83 x 10(5) cells/cm2 on foams with 300-500 microns pores resulted in a cell density of 4.63 x 10(5) cells/cm2 after 1 day in culture; they had alkaline phosphatase activities of 4.28 x 10(-7) and 2.91 x 10(-6) mumol/cell/min on Days 7 and 28, respectively; and they had a cell density that increased to 18.7 x 10(5) cells/cm2 by Day 56. For the same constructs, the mineralized matrix reached a maximum penetration depth of 240 microns from the top surface of the foam and a value of 0.083 mm for mineralized tissue volume per unit of cross sectional area. Seeding density was an important parameter for the constructs, but pore size over the range tested did not affect cell proliferation or function. This study suggests the feasibility of using poly(alpha-hydroxy ester) foams as scaffolding materials for the transplantation of autogenous osteoblasts to regenerate bone tissue.

  3. Adipogenic RNAs are transferred in osteoblasts via bone marrow adipocytes-derived extracellular vesicles (EVs).

    PubMed

    Martin, Perrine J; Haren, Nathalie; Ghali, Olfa; Clabaut, Aline; Chauveau, Christophe; Hardouin, Pierre; Broux, Odile

    2015-03-18

    In osteoporosis, bone loss is accompanied by increased marrow adiposity. Given their proximity in the bone marrow and their shared origin, a dialogue between adipocytes and osteoblasts could be a factor in the competition between human Mesenchymal Stem Cells (hMSC) differentiation routes, leading to adipocyte differentiation at the expense of osteoblast differentiation. The adipocyte/osteoblast balance is highly regulated at the level of gene transcription. In our work, we focused on PPARgamma, CEBPalpha and CEBPdelta, as these transcription factors are seen as master regulators of adipogenesis and expressed precociously, and on leptin and adiponectin, considered as adipocyte marker genes. In 2010, our group has demonstrated, thanks to a coculture model, that in the presence of hMSC-derived adipocytes (hMSC-Adi), hMSC-derived osteoblasts (hMSC-Ost) express lesser amounts of osteogenic markers but exhibit the expression of typical adipogenic genes. Nevertheless, the mechanisms underlying this modulation of gene expression are not clarified. Recently, adipocytes were described as releasing extracellular vesicles (EVs), containing and transferring adipocyte specific transcripts, like PPARgamma, leptin and adiponectin. Here, we investigated whether EVs could be the way in which adipocytes transfer adipogenic RNAs in our coculture model. We observed in hMSC-Ost incubated in hAdi-CM an increase in the adipogenic PPARγ, leptin, CEBPα and CEBPδ transcripts as well as the anti-osteoblastic miR-138, miR30c, miR125a, miR-125b, miR-31 miRNAs, probably implicated in the observed osteocalcin (OC) and osteopontin (OP) expression decrease. Moreover, EVs were isolated from conditioned media collected from cultures of hMSC at different stages of adipocyte differentiation and these specific adipogenic transcripts were detected inside. Finally, thanks to interspecies conditioned media exposition, we could highlight for the first time a horizontal transfer of adipogenic transcripts

  4. Tumour necrosis factor-alpha (TNFα) stimulates the growth of human bone marrow stromal cells

    PubMed Central

    Rougier, F.; Cornu, E.; Gachard, N.; Praloran, V.

    1997-01-01

    This study reports that TNF-α is a potent mitogen for human bone marrow sternal cells in vitro (assessed by [3H]-thymidine incorporation into DNA and cell counts). In contrast, cytokines such as IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, LIF, SCF, M-CSF, G-CSF and GM-CSF had no effect. The effect of TNF-α on the growth of human bone marrow stromal cells could be of importance during inflammatory processes which take place in the marrow, for example marrow fibrosis. PMID:18472825

  5. Glycosaminoglycans enhance osteoblast differentiation of bone marrow derived human mesenchymal stem cells.

    PubMed

    Mathews, Smitha; Mathew, Suja Ann; Gupta, Pawan Kumar; Bhonde, Ramesh; Totey, Satish

    2014-02-01

    Extracellular matrix plays an important role in regulating cell growth and differentiation. The biomimetic approach of cell-based tissue engineering is based on mirroring this in vivo micro environment for developing a functional tissue engineered construct. In this study, we treated normal tissue culture plates with selected extracellular matrix components consisting of glycosaminoglycans such as chondroitin-4-sulphate, dermatan sulphate, chondroitin-6-sulphate, heparin and hyaluronic acid. Mesenchymal stem cells isolated from adult human bone marrow were cultured on the glycosaminoglycan treated culture plates to evaluate their regulatory role in cell growth and osteoblast differentiation. Although no significant improvement on human mesenchymal stem cell adhesion and proliferation was observed on the glycosaminoglycan-treated tissue culture plates, there was selective osteoblast differentiation, indicating its potential role in differentiation rather than proliferation. Osteoblast differentiation studies showed high osteogenic potential for all tested glycosaminoglycans except chondroitin-4-sulphate. Osteoblast differentiation-associated genes such as osterix, osteocalcin, integrin binding sialoprotein, osteonectin and collagen, type 1, alpha 1 showed significant upregulation. We identified osterix as the key transcription factor responsible for the enhanced bone matrix deposition observed on hyaluronic acid, heparin and chondroitin-6-sulphate. Hyaluronic acid provided the most favourable condition for osteoblast differentiation and bone matrix synthesis. Our results confirm and emphasise the significant role of extracellular matrix in regulating cell differentiation. To summarise, glycosaminoglycans of extracellular matrix played a significant role in regulating osteoblast differentiation and could be exploited in the biomimetic approach of fabricating or functionalizing scaffolds for stem cell based bone tissue engineering.

  6. Stromal cells in long-term murine bone marrow culture: FACS studies and origin of stromal cells in radiation chimeras

    SciTech Connect

    Lennon, J.E.; Micklem, H.S.

    1986-05-01

    Adherent layers from hematopoietically active long-term bone marrow cultures (LTBMC), incubated with fluorescent beads, were analyzed for autofluorescence and phagocytic ability, using a fluorescence-activated cell sorter (FACS). Four groups of cells were separated from the adherent layers, including a group of large polygonal fibroblastoid stromal cells. Long-term chimeras were made by lethal irradiation of CBA/Ca (CBA) and C57Bl6/J (B6) mice and repopulation with phosphoglycerate kinase (PGK-1) alloenzyme-congenic bone marrow cells. Hematopoietically active LTBMC were established from such chimeras, and donor and host contributions of FACS-sorted adherent-layer cells were measured. While macrophages and other hematopoietic cells were of donor origin, the fibroblastoid stromal cells were mainly or entirely host derived.

  7. Effect of dexamethasone on moesin gene expression in rabbit bone marrow stromal cells.

    PubMed

    Cornet, F; Broux, O; Anselme, K; Hardouin, P; Jeanfils, J

    2004-10-01

    The influence of dexamethasone on rabbit bone marrow stromal cells differentiation was studied by screening the action of dexamethasone on gene expression. Using differential display, we observed some differential amplifications. The use of five of thirteen different primers combination allowed to identify one or more differential bands. One of them was identified as moesin gene. Real-time PCR confirmed a significant reduction of moesin gene expression following dexamethasone treatment. The decrease of expression for this protein, involved in cytoskeletal organization, could explain the effects of dexamethasone treatment on bone marrow stromal cells differentiation.

  8. Bone Marrow Stromal Stem Cells in Tissue Engineering and Regenerative Medicine.

    PubMed

    Polymeri, A; Giannobile, W V; Kaigler, D

    2016-11-01

    Bone marrow stromal stem cells (BMSCs) are adult multipotent cells, which have the potential to differentiate into cell types of mesodermal origin, namely osteocytes, adipocytes, and chondrocytes. Due to their accessibility and expansion potential, BMSCs have historically held therapeutic promise in tissue engineering and regenerative medicine applications. More recently, it has been demonstrated that not only can bone marrow stromal stem cells directly participate in tissue regeneration, but they also have the capacity to migrate to distant sites of tissue injury, where they can participate in tissue repair either directly through their differentiation or indirectly through paracrine mechanisms. Additionally, they can elicit various immunomodulatory signals, which can attenuate the inflammatory and immune responses. As such, bone marrow stromal stem cells have been explored clinically for treatment of a wide variety of different conditions including bone defects, graft-vs.-host disease, cardiovascular diseases, autoimmune diseases, diabetes, neurological diseases, and liver and kidney diseases. This review provides an overview of current clinical applications of bone marrow stromal stem cells and discusses their therapeutic properties, while also addressing limitations of their use. PubMed, Ovid, and Google Scholar online databases were searched using several keywords, including "stem cells", "tissue engineering", tissue regeneration" and "clinical trials". Additionally, Clinical trials.gov was used to locate completed clinical trials using bone marrow derived stem cells.

  9. Extracellular inorganic phosphate regulates gibbon ape leukemia virus receptor-2/phosphate transporter mRNA expression in rat bone marrow stromal cells.

    PubMed

    Wada, Keinoshin; Mizuno, Morimichi; Komori, Takahide; Tamura, Masato

    2004-01-01

    In mammalian cells, several observations indicate not only that phosphate transport probably regulates local inorganic phosphate (Pi) concentration, but also that Pi affects normal cellular metabolism, which in turn regulates apoptosis and the process of mineralization. To elucidate how extracellular Pi regulates cellular functions of pre-osteoblastic cells, we investigated the expression of type III sodium (Na)-dependent Pi transporters in rat bone marrow stromal cells and ROB-C26 pre-osteoblastic cells. The mRNA expression level of gibbon ape leukemia virus receptor (Glvr)-2 was increased by the addition of Pi in rat bone marrow stromal cells, but not in ROB-C26 or normal rat kidney (NRK) cells. In contrast, the level of Glvr-1 mRNA was not altered by the addition of extracellular Pi in these cells. The induction of Glvr-2 mRNA by Pi was inhibited in the presence of cycloheximide (CHX). Moreover, mitogen-activated protein kinase (MEK) /extracellular-signal-regulated kinase (ERK) pathway inhibitors; U0126 (1.4-diamino-2, 3-dicyano-1, 4-bis [2-amino-phenylthio] butadiene) and PD98059 (2'-Amino-3'-methoxyflavone) inhibited inducible Glvr-2 mRNA expression, but p38 MEK inhibitor SB203580 [4-(4'-fluorophenyl)-2-(4'-methyl-sulfinylphenyl)-5-(4'pyridyl) imidazole] did not inhibit the induction of Glvr-2 mRNA expression, suggesting that extracellular Pi regulates de novo protein synthesis and MEK/ERK activity in rat bone marrow stromal cells, and through these, induction of Glvr-2 mRNA. Although Pi also induced osteopontin mRNA expression in rat bone marrow stromal cells but not in ROB-C26 and NRK cells, changes in cell viability with the addition of Pi were similar in both cell types. These data indicate that extracellular Pi regulates Glvr-2 mRNA expression, provide insights into possible mechanisms whereby Pi may regulate protein phosphorylation, and suggest a potential role for the Pi transporter in rat bone marrow stromal cells.

  10. Cell attachment and proliferation of bone marrow-derived osteoblast on zirconia of various surface treatment

    PubMed Central

    Lee, Heesu; Noh, Kwantae; Woo, Yi-Hyung

    2014-01-01

    PURPOSE This study was performed to characterize the effects of zirconia coated with calcium phosphate and hydroxyapatite compared to smooth zirconia after bone marrow-derived osteoblast culture. MATERIALS AND METHODS Bone marrow-derived osteoblasts were cultured on (1) smooth zirconia, (2) zirconia coated with calcium phosphate (CaP), and (3) zirconia coated with hydroxyapatite (HA). The tetrazolium-based colorimetric assay (MTT test) was used for cell proliferation evaluation. Scanning electron microscopy (SEM) and alkaline phosphatase (ALP) activity was measured to evaluate the cellular morphology and differentiation rate. X-ray photoelectron spectroscopy (XPS) was employed for the analysis of surface chemistry. The genetic expression of the osteoblasts and dissolution behavior of the coatings were observed. Assessment of the significance level of the differences between the groups was done with analysis of variance (ANOVA). RESULTS From the MTT assay, no significant difference between smooth and surface coated zirconia was found (P>.05). From the SEM image, cells on all three groups of discs were sporadically triangular or spread out in shape with formation of filopodia. From the ALP activity assay, the optical density of osteoblasts on smooth zirconia discs was higher than that on surface treated zirconia discs (P>.05). Most of the genes related to cell adhesion showed similar expression level between smooth and surface treated zirconia. The dissolution rate was higher with CaP than HA coating. CONCLUSION The attachment and growth behavior of bone-marrow-derived osteoblasts cultured on smooth surface coated zirconia showed comparable results. However, the HA coating showed more time-dependent stability compared to the CaP coating. PMID:24843393

  11. ATP release mediates fluid flow-induced proliferation of human bone marrow stromal cells.

    PubMed

    Riddle, Ryan C; Taylor, Amanda F; Rogers, Jennifer R; Donahue, Henry J

    2007-04-01

    Oscillatory fluid flow induced the vesicular release of ATP from human BMSCs that directly contributes to the induction of BMSC proliferation. Degrading extracellular nucleotides prevents fluid flow-induced increases in intracellular calcium concentration, the activation of calcineurin, and the nuclear translocation of NFAT. Regulation of bone cell activity by autocrine/paracrine factors is a well-established mechanism by which skeletal homeostasis is regulated by mechanical signals. The release of extracellular nucleotides in particular has been shown to induce many of the responses thought to be necessary for load-induced bone formation. In these studies, we examined the effect of oscillatory fluid flow on the release of ATP from bone marrow stromal cells (BMSCs) and the effect of ATP release on BMSC proliferation and intracellular calcium signaling pathways. BMSCs were exposed to oscillatory fluid flow, and the concentration of ATP in conditioned media samples was determined using a luciferin:luciferase-based reaction. Western blot analysis was used to examine the expression of purinergic receptors. Using pharmacological antagonists of gap junction hemichannels and vesicular trafficking, we studied the mechanism of ATP release from BMSCs. Apyrase was used to study the effect of extracellular nucleotides on intracellular calcium concentration, calcineurin activity, and nuclear factor of activated T cells (NFAT) nuclear translocation. Fluid flow exposure induced the flow rate-dependent release of ATP from BMSCs that was attenuated by treatment with monensin and N-ethylmaleimide, suggesting a vesicular mechanism. Treating BMSCs with ATP, but not other nucleotides, increased cellular proliferation. Moreover, extracellular ATP was a prerequisite for fluid flow-induced increases in intracellular calcium concentration, activation of calcineurin, the nuclear translocation of NFATc1, and proliferation. These data indicate that ATP regulates not only osteoblastic and

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

    PubMed

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

    2011-07-01

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

  13. Dexamethasone Regulates EphA5, a Potential Inhibitory Factor with Osteogenic Capability of Human Bone Marrow Stromal Cells

    PubMed Central

    Yamada, Tsuyoshi; Yoshii, Toshitaka; Yasuda, Hiroaki; Okawa, Atsushi; Sotome, Shinichi

    2016-01-01

    We previously demonstrated the importance of quality management procedures for the handling of human bone marrow stromal cells (hBMSCs) and provided evidence for the existence of osteogenic inhibitor molecules in BMSCs. One candidate inhibitor is the ephrin type-A receptor 5 (EphA5), which is expressed in hBMSCs and upregulated during long-term culture. In this study, forced expression of EphA5 diminished the expression of osteoblast phenotypic markers. Downregulation of endogenous EphA5 by dexamethasone treatment promoted osteoblast marker expression. EphA5 could be involved in the normal growth regulation of BMSCs and could be a potential marker for replicative senescence. Although Eph forward signaling stimulated by ephrin-B-Fc promoted the expression of ALP mRNA in BMSCs, exogenous addition of EphA5-Fc did not affect the ALP level. The mechanism underlying the silencing of EphA5 in early cultures remains unclear. EphA5 promoter was barely methylated in hBMSCs while histone deacetylation could partially suppress EphA5 expression in early-passage cultures. In repeatedly passaged cultures, the upregulation of EphA5 independent of methylation could competitively inhibit osteogenic signal transduction pathways such as EphB forward signaling. Elucidation of the potential inhibitory function of EphA5 in hBMSCs may provide an alternative approach for lineage differentiation in cell therapy strategies and regenerative medicine. PMID:27057165

  14. Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation

    PubMed Central

    Hilton, Matthew J.; Tu, Xiaolin; Wu, Ximei; Bai, Shuting; Zhao, Haibo; Kobayashi, Tatsuya; Kronenberg, Henry M.; Teitelbaum, Steven L.; Ross, F. Patrick; Kopan, Raphael; Long, Fanxin

    2009-01-01

    Postnatal bone marrow houses mesenchymal progenitor cells that are osteoblast precursors. These cells have established therapeutic potential 1 but they are difficult to maintain and expand in vitro, presumably because little is known about the mechanisms controlling their fate decisions. To investigate the potential role of Notch signaling in osteoblastogenesis, we used conditional alleles to genetically remove components of the Notch signaling system during skeletal development. We find that Notch disruption in the limb skeletogenic mesenchyme markedly enhanced trabecular bone mass in adolescent mice. Notably, mesenchymal progenitors were virtually depleted in the bone marrow of the high-bone-mass animals. As a result, these animals developed severe osteopenia as they aged. Moreover, Notch appeared to inhibit osteoblast differentiation through Hes/Hey proteins that diminished Runx2 transcriptional activity via physical interaction. These results support a model wherein Notch signaling in bone marrow normally acts to maintain a pool of mesenchymal progenitors by suppressing osteoblast differentiation. Thus, mesechymal progenitors may be expanded in vitro by activating Notch, whereas bone formation in vivo may be enhanced by transiently suppressing this pathway. PMID:18297083

  15. Osteoblast-specific gene expression after transplantation of marrow cells: Implications for skeletal gene therapy

    PubMed Central

    Hou, Zhen; Nguyen, Que; Frenkel, Baruch; Nilsson, Susan K.; Milne, Moira; van Wijnen, André J.; Stein, Janet L.; Quesenberry, Peter; Lian, Jane B.; Stein, Gary S.

    1999-01-01

    Somatic gene therapies require targeted transfer of the therapeutic gene(s) into stem cells that proliferate and then differentiate and express the gene in a tissue-restricted manner. We have developed an approach for gene therapy using marrow cells that takes advantage of the osteoblast specificity of the osteocalcin promoter to confine expression of chimeric genes to bone. Adherent marrow cells, carrying a reporter gene [chloramphenicol acetyltransferase (CAT)] under the control of a 1.7-kilobase rat osteocalcin gene promoter, were expanded ex vivo. After transplantation by intravenous infusion, engrafted donor cells in recipient mice were detected by the presence of the transgene in a broad spectrum of tissues. However, expression of the transgene was restricted to osteoblasts and osteocytes, as established by biochemical analysis of CAT activity and immunohistochemical analysis of CAT expression at the single cell level. Our data indicate that donor cells achieved long-term engraftment in various tissues of the recipients and that the CAT gene under control of the osteocalcin promoter is expressed specifically in bone. Thus, transplantation of multipotential marrow cells containing the osteocalcin promoter-controlled transgene provides an efficacious approach to deliver therapeutic gene expression to osteoblasts for treatment of bone disorders or tumor metastasis to the skeleton. PMID:10377408

  16. ERR{alpha} regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells

    SciTech Connect

    Rajalin, Ann-Marie; Pollock, Hanna; Aarnisalo, Piia

    2010-05-28

    The orphan nuclear receptor estrogen-related receptor-{alpha} (ERR{alpha}) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERR{alpha} in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERR{alpha} deficient mice and their differentiation capacities were compared to that of the wild-type cells. ERR{alpha} deficient cultures displayed reduced cellular proliferation, osteoblastic differentiation, and mineralization. In the complementary experiment, overexpression of ERR{alpha} in MC3T3-E1 cells increased the expression of osteoblastic markers and mineralization. Alterations in the expression of bone sialoprotein (BSP) may at least partially explain the effects on mineralization as BSP expression was reduced in ERR{alpha} deficient MSCs and enhanced upon ERR{alpha} overexpression in MC3T3-E1 cells. Furthermore, a luciferase reporter construct driven by the BSP promoter was efficiently transactivated by ERR{alpha}. Under adipogenic conditions, ERR{alpha} deficient cultures displayed reduced adipocytic differentiation. Our data thus propose a positive role for ERR{alpha} in osteoblastic and adipocytic differentiation. The variability in the results yielded in the different studies implies that ERR{alpha} may play different roles in bone under different physiological conditions.

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

    PubMed Central

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

    2017-01-01

    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 κ-B ligan, and pre-collagen type 1 α 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 κ-B ligand, and pre-collagen type 1 α 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. PMID:28133520

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

    PubMed

    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. Effects of arachidonic acid on the concentration of hydroxyeicosatetraenoic acids in culture media of mesenchymal stromal cells differentiating into adipocytes or osteoblasts.

    PubMed

    Casado-Díaz, Antonio; Ferreiro-Vera, Carlos; Priego-Capote, Feliciano; Dorado, Gabriel; Luque-de-Castro, María Dolores; Quesada-Gómez, José Manuel

    2014-01-01

    Metabolites derived from the polyunsaturated fatty acids (PUFA) may modulate the mesenchymal stromal cell (MSC) differentiation. Such cells can differentiate into different cellular types, including adipocytes and osteoblasts. Aging favors the bone marrow MSC differentiation toward the former, causing a loss of bone density associated with pathologies like osteoporosis. The omega-6 arachidonic acid (AA) favors MSC adipogenesis to a greater extent than omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In this work, we study the joint action of both PUFA. Thus, not induced and induced to adipocyte or osteoblast MSC were treated with 20 μM of each PUFA (either AA, AA + DHA or AA + EPA). The expression of osteogenic and adipogenic molecular markers, the alox15b lipoxygenase gene expression and the 5-, 8-, 11-, 12- and 15-hydroxyeicosatetraenoic acids (HETE) derived from the AA metabolism in the culture media were determined. The results show that the adipogenesis induction of AA is not suppressed by the joint presence of EPA and DHA. In fact, both increased the adipogenic effect of AA on MSC differentiated into osteoblasts. The different HETE concentrations increased in cultures supplemented with AA, albeit such concentrations were lower in the cultures induced to differentiate, mainly at day 21 after the induction. Furthermore, the reduction in the HETE concentration was correlated with a higher expression of the alox15b gene. These results highlight the PUFA metabolism differences between uninduced and induced MSC to differentiate into adipocytes and osteoblasts, besides the relevant role of the lipoxygenase gene expression in adipogenesis induction.

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

    PubMed

    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.

  1. Strain energy density gradients in bone marrow predict osteoblast and osteoclast activity: a finite element study.

    PubMed

    Webster, Duncan; Schulte, Friederike A; Lambers, Floor M; Kuhn, Gisela; Müller, Ralph

    2015-03-18

    Huiskes et al. hypothesized that mechanical strains sensed by osteocytes residing in trabecular bone dictate the magnitude of load-induced bone formation. More recently, the mechanical environment in bone marrow has also been implicated in bone׳s response to mechanical stimulation. In this study, we hypothesize that trabecular load-induced bone formation can be predicted by mechanical signals derived from an integrative µFE model, incorporating a description of both the bone and marrow phase. Using the mouse tail loading model in combination with in vivo micro-computed tomography (µCT) we tracked load induced changes in the sixth caudal vertebrae of C57BL/6 mice to quantify the amount of newly mineralized and eroded bone volumes. To identify the mechanical signals responsible for adaptation, local morphometric changes were compared to micro-finite element (µFE) models of vertebrae prior to loading. The mechanical parameters calculated were strain energy density (SED) on trabeculae at bone forming and resorbing surfaces, SED in the marrow at the boundary between bone forming and resorbing surfaces, along with SED in the trabecular bone and marrow volumes. The gradients of each parameter were also calculated. Simple regression analysis showed mean SED gradients in the trabecular bone matrix to significantly correlate with newly mineralized and eroded bone volumes R(2)=0.57 and 0.41, respectively, p<0.001). Nevertheless, SED gradients in the marrow were shown to be the best predictor of osteoblastic and osteoclastic activity (R(2)=0.83 and 0.60, respectively, p<0.001). These data suggest that the mechanical environment of the bone marrow plays a significant role in determining osteoblast and osteoclast activity.

  2. Stromal cells from human long-term marrow cultures, but not cultured marrow fibroblasts, phagocytose horse serum constituents: studies with a monoclonal antibody that reacts with a species-specific epitope common to multiple horse serum proteins.

    PubMed

    Charbord, P; Tippens, D; Wight, T S; Gown, A M; Singer, J W

    1987-01-01

    This report describes an IgG1 mouse monoclonal antibody derived after immunization of mice with washed stromal cells from human, long-term bone marrow cultures. The antigen recognized by the antibody (BMS-1) is a carbohydrate-containing prosthetic group that is common to and specific for multiple horse serum proteins. These proteins are avidly ingested by stromal cells and concentrated in endocytic vesicles. Cultured smooth muscle cells took up the horse proteins in a similar manner to marrow stromal cells while cultured marrow fibroblasts, endothelial cells, and hepatoma cells did not. These data indicate that marrow stromal cells specifically accumulate horse serum proteins which might partially explain the horse serum requirement for long-term marrow culture maintenance. The data also suggest further similarities between marrow stromal and smooth muscle cells and additional differences between marrow fibroblasts and marrow stromal cells.

  3. Bone Regeneration Induced by Bone Porcine Block with Bone Marrow Stromal Stem Cells in a Minipig Model of Mandibular “Critical Size” Defect

    PubMed Central

    Di Benedetto, Adriana; Cozzolino, Valerio; Podaliri Vulpiani, Michele; Grano, Maria; Kalemaj, Zamira; Grassi, Felice Roberto

    2017-01-01

    Introduction. Adding stem cells to biodegradable scaffolds to enhance bone regeneration is a valuable option. Different kinds of stem cells with osteoblastic activity were tested, such as bone marrow stromal stem cells (BMSSCs). Aim. To assess a correct protocol for osteogenic stem cell differentiation, so BMSSCs were seeded on a bone porcine block (BPB). Materials and Methods. Bone marrow from six minipigs was extracted from tibiae and humeri and treated to isolate BMSSCs. After seeding on BPB, critical-size defects were created on each mandible of the minipigs and implanted with BPB and BPB/BMSSCs. After three months, histomorphometric analysis was performed. Results. Histomorphometric analysis provided percentages of the three groups. Tissues present in control defects were 23 ± 2% lamellar bone, 28 ± 1% woven bone, and 56 ± 4% marrow spaces; in BPB defects were 20 ± 5% BPB, 32 ± 2% lamellar bone, 24 ± 1% woven bone, and 28 ± 2% marrow spaces; in BPB/BMSSCs defects were 17 ± 4% BPB/BMSSCs, 42 ± 2% lamellar bone, 12 ± 1% woven bone, and 22 ± 3% marrow spaces. Conclusion. BPB used as a scaffold to induce bone regeneration may benefit from the addition of BDPSCs in the tissue-engineered constructs. PMID:28553359

  4. Disruption of the Fgf2 Gene Activates the Adipogenic and Suppresses the Osteogenic Program in Mesenchymal Marrow Stromal Stem Cells

    PubMed Central

    Xiao, Liping; Sobue, Takanori; Eisliger, Alycia; Kronenberg, Mark. S; Coffin, J. Douglas; Doetschman, Thomas; Hurley, Marja M.

    2010-01-01

    Here we determine the Fibroblast Growth Factor-2 (FGF2) dependency of the time course of changes in bone mass in female mice. This study extends our earlier reports that knockout of the FGF2 gene (Fgf2) caused low turnover bone loss in Fgf2−/− male mice by examining bone loss with age in Fgf2−/− female mice, and by assessing whether reduced bone formation is associated with differentiation of bone marrow stromal cells (BMSCs) towards the adipocyte lineage. Bone mineral density (BMD) was similar in 3 month old female Fgf2+/+ and Fgf2−/− mice but was significantly reduced as early as 5 months of age in Fgf2−/− mice. In vivo studies showed that there was a greater accumulation of marrow fat in long bones of 14 and 20 month old Fgf2−/− mice compared with Fgf2+/+ littermates. To study the effect of disruption of FGF2 on osteoblastogenesis and adipogenesis, BMSCs from both genotypes were cultured in osteogenic or adipogenic media. Reduced alkaline phosphatase positive (ALP), mineralized colonies and a marked increase in adipocytes were observed in Fgf2−/− BMSC cultures. These cultures also showed an increase in the mRNA of the adipogenic transcription factor PPARγ2 as well as the downstream target genes aP2 and adiponectin. Treatment with exogenous FGF2 blocked adipocyte formation and increased ALP colony formation and ALP activity in BMSC cultures of both genotypes. These results support an important role for endogenous FGF2 in osteoblast (OB) lineage determination. Alteration in FGF2 signaling may contribute to impaired OB bone formation capacity and to increased bone marrow fat accumulation both of which are characteristics of aged bone. PMID:20510392

  5. Mesenchymal stromal cells from bone marrow treated with bovine tendon extract acquire the phenotype of mature tenocytes☆

    PubMed Central

    Augusto, Lívia Maria Mendonça; Aguiar, Diego Pinheiro; Bonfim, Danielle Cabral; dos Santos Cavalcanti, Amanda; Casado, Priscila Ladeira; Duarte, Maria Eugênia Leite

    2016-01-01

    Objective This study evaluated in vitro differentiation of mesenchymal stromal cells isolated from bone marrow, in tenocytes after treatment with bovine tendon extract. Methods Bovine tendons were used for preparation of the extract and were stored at −80 °C. Mesenchymal stromal cells from the bone marrow of three donors were used for cytotoxicity tests by means of MTT and cell differentiation by means of qPCR. Results The data showed that mesenchymal stromal cells from bone marrow treated for up to 21 days in the presence of bovine tendon extract diluted at diminishing concentrations (1:10, 1:50 and 1:250) promoted activation of biglycan, collagen type I and fibromodulin expression. Conclusion Our results show that bovine tendon extract is capable of promoting differentiation of bone marrow stromal cells in tenocytes. PMID:26962503

  6. Effectiveness of Bone Marrow Stromal Cell Sheets in Maintaining Random-Pattern Skin Flaps in an Experimental Animal Model.

    PubMed

    Kira, Tsutomu; Omokawa, Shohei; Akahane, Manabu; Shimizu, Takamasa; Nakano, Kenichi; Nakanishi, Yasuaki; Onishi, Tadanobu; Kido, Akira; Inagaki, Yusuke; Tanaka, Yasuhito

    2015-11-01

    Bone marrow stromal cells can be applied therapeutically to enhance angiogenesis; however, the use of bone marrow stromal cell suspensions reduces efficiency because of low-level attachment. The authors hypothesized that bone marrow stromal cell sheets would facilitate cell fixation, thus enhancing angiogenesis. The authors investigated flap survival area and enhancement of angiogenic factors in a rat random-pattern skin flap model after application of bone marrow stromal cell sheets. Bone marrow stromal cell sheets (prepared from 7-week-old rat femurs) were cultured under four different hypoxic conditions. Sheets with the highest angiogenic potential, determined by an in vitro pilot study, were injected into subcutaneous layers of the rat dorsum (bone marrow stromal cell sheet group). A control group (phosphate-buffered saline only) was included. On day 2 after injection, caudally based random-pattern skin flaps (12 × 3 cm) were elevated. On day 7 after elevation, surviving skin flap areas were measured. Skin samples were harvested from each flap and gene expression levels of vascular endothelial growth factor and basic fibroblast growth factor were measured by quantitative real-time polymerase chain reaction. Skin flap survival area (71.6 ± 2.3 percent versus 51.5 ± 3.3 percent) and levels of vascular endothelial growth factor and basic fibroblast growth factor were significantly higher in the bone marrow stromal cell sheet group than in the control group (p < 0.05). Implantation of bone marrow stromal cell sheets increased the survival area of random-pattern skin flaps. Expression of angiogenic factors may have contributed to the increased flap survival.

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

    NASA Astrophysics Data System (ADS)

    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.

  8. Promoting spinal fusions by biomineralized silk fibroin films seeded with bone marrow stromal cells: An in vivo animal study.

    PubMed

    Gu, Yong; Chen, Liang; Niu, Hai-Yun; Shen, Xiao-Feng; Yang, Hui-Lin

    2016-03-01

    To prepare a biomineralized nano silk fibroin film seeded with bone marrow stromal cells (BMSCs), and to evaluate its performance in spinal fusion. The silk fibroin film was mineralized in a modified, simulated body fluid, seeded with BMSCs, and evaluated in a rat model of posterolateral lumbar fusion, compared with pure silk fibroin, silk fibroin/bone marrow stromal cells, mineralized silk fibroin, mineralized silk fibroin/bone marrow stromal cells, iliac crest bone, and no graft. After 12 weeks, all rats were sacrificed and underwent manual palpation, micro-CT scanning, biomechanical testing, and histology. The infrared spectrum, X-ray diffraction, and scanning electron microscopy demonstrated deposition of mineral layers on the silk fibroin film surface. The fusion rate, bone volume, relative strength and stiffness, and histological score of the mineralized silk fibroin/bone marrow stromal cells were slightly lower than the autograft, but without any significant difference (p > 0.05). In addition, the mineralized silk fibroin was significantly greater in most parameters than the silk fibroin/bone marrow stromal cells (p < 0.05). The mineralized silk fibroin resembles natural bone structurally, and the cellular and mineral layers of silk fibroin are both critical to bone regeneration. The ability to promote spinal fusion is enhanced when the mineralized silk fibroin is seeded with bone marrow stromal cells. © The Author(s) 2015.

  9. Bone marrow-derived stromal cells are associated with gastric cancer progression

    PubMed Central

    Kasashima, H; Yashiro, M; Nakamae, H; Masuda, G; Kinoshita, H; Morisaki, T; Fukuoka, T; Hasegawa, T; Sakurai, K; Toyokawa, T; Kubo, N; Tanaka, H; Muguruma, K; Ohira, M; Nakane, T; Hino, M; Hirakawa, K

    2015-01-01

    Background: The aim of this study was to clarify the role of bone marrow-derived stromal cells (BM-SCs) expressing CD271 in the development of gastric cancer. Methods: The effect of human BM-SCs on the proliferation and motility of six gastric cancer cell lines, OCUM-2M, OCUM-2MD3, OCUM-12, KATO-III, NUGC-3, and MKN-74, was examined. CD271 expression levels in BM-SCs were analysed by flow cytometry. We also generated a gastric tumour model by orthotopic inoculation of OCUM-2MLN cells in mice that had received transplantation of bone marrow from the CAG-EGFP mice. The correlation between the clinicopathological features of 279 primary gastric carcinomas and CD271 expression in tumour stroma was examined by immunohistochemistry. Results: Numerous BM-SCs infiltrated the gastric tumour microenvironment; CD271 expression was found in ∼25% of BM-SCs. Conditioned medium from BM-SCs significantly increased the proliferation of gastric cancer cell lines. Furthermore, conditioned medium from gastric cancer cells significantly increased the number of BM-SCs, whereas migration of OCUM-12 and NUGC-3 cells was significantly increased by conditioned medium from BM-SCs. CD271 expression in stromal cells was significantly associated with macroscopic type-4 cancers, diffuse-type tumours, and tumour invasion depth. The overall survival of patients (n=279) with CD271-positive stromal cells was significantly worse compared with that of patients with CD271-negative stromal cells. This is the first report of the significance of BM-SCs in gastric cancer progression. Conclusions: Bone marrow-derived stromal cells might have an important role in gastric cancer progression, and CD271-positive BM-SCs might be a useful prognostic factor for gastric cancer patients. PMID:26125445

  10. Ultrastructural localization of stem cell factor in canine marrow-derived stromal cells.

    PubMed

    Huss, R; Hong, D S; Beckham, C; Kimball, L; Myerson, D H; Storb, R; Deeg, H J

    1995-01-01

    Stromal cell lines derived from canine long-term bone marrow cultures (LTBMC) were characterized regarding the expression of growth factors and especially the localization of stem cell factor (SCF) (c-kit ligand). One cell line (DO64) was immortalized by transformation with a retroviral vector containing the open reading frames (ORFs) E6 and E7 of the human papilloma virus type 16 (HPV-16). Transfection did not change cellular characteristics but rendered the cell line more independent from culture conditions. The transformed line DO64 consisted mainly of fibroblast-like cells. In addition, some cells showed endothelial and some smooth-muscle cell features. Stromal cells expressed a broad spectrum of surface markers, including low levels of major histocompatibility-complex (MHC) class-II antigens. A new murine monoclonal antibody (MAb), RG7.6 (IgG1), specific for canine SCF, recognized the majority of fibroblast-like stromal cells. The staining pattern for SCF showed perinuclear and intracytoplasmic dense areas. Immunoelectron microscopy revealed the localization of SCF in secretory vesicles, the perivesicular cytoplasm, and bound to the cytoplasmatic membrane. RNA analysis showed that stromal cells transcribed, in addition to SCF, messages for granulocyte colony-stimulating factor (G-CSF), granulocyte-monocyte CSF (GM-CSF), interleukin-6 (IL-6), and transforming growth factor-beta (TGF-beta). In summary, we have established and characterized canine marrow-derived stromal cell lines, and using the new MAb RG7.6, we have localized SCF to cytoplasmatic vesicles as well as the membrane of stromal cells.

  11. Notch signalling drives bone marrow stromal cell-mediated chemoresistance in acute myeloid leukemia.

    PubMed

    Takam Kamga, Paul; Bassi, Giulio; Cassaro, Adriana; Midolo, Martina; Di Trapani, Mariano; Gatti, Alessandro; Carusone, Roberta; Resci, Federica; Perbellini, Omar; Gottardi, Michele; Bonifacio, Massimiliano; Nwabo Kamdje, Armel Hervé; Ambrosetti, Achille; Krampera, Mauro

    2016-04-19

    Both preclinical and clinical investigations suggest that Notch signalling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms. However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-κB.These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML.

  12. Suppression of PPAR transactivation switches cell fate of bone marrow stem cells from adipocytes into osteoblasts.

    PubMed

    Takada, Ichiro; Suzawa, Miyuki; Matsumoto, Kunihiro; Kato, Shigeaki

    2007-11-01

    Osteoblasts and adipocytes differentiate from common pleiotropic mesenchymal stem cells under transcriptional controls by numerous factors and multiple intracellular signalings. However, cellular signaling factors that determine cell fates of mensenchymal stem cells in bone marrow remain to be largely uncovered, though peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is well established as a prime inducer of adipogenesis. Here, we describe two signaling pathways that induce the cell fate decision into osteoblasts from adipocytes. One signaling is a TAK1/TAB1/NIK cascade activated by TNF-alpha and IL-1, and the activated NF-kappaB blocked the DNA binding of PPAR-gamma, attenuating the activated PPAR-mediated adipogenesis. The second signaling is the noncanonical Wnt pathway through CaMKII-TAK1/TAB2-NLK. Activated NLK by a noncanonical Wnt ligand (Wnt-5a) transrepresses PPAR transactivation through a histone methyltransferase, SETDB1. Wnt-5a induces phosphorylation of NLK, leading to the formation of a corepressor complex that inactivates PPAR function through histone H3-K9 methylation. Thus, two signaling pathways lead to an osteoblastic cell lineage decision from mesenchymal stem cells through two distinct modes of PPAR transrepression.

  13. Glycitin regulates osteoblasts through TGF-β or AKT signaling pathways in bone marrow stem cells

    PubMed Central

    Zhang, Liyan; Chen, Jiying; Chai, Wei; Ni, Min; Sun, Xin; Tian, Dan

    2016-01-01

    The aim of the present study was to examine the effect of glycitin on the regulation of osteoblasts from bone marrow stem cells (BMSCs) through transforming growth factor (TGF)-β or protein kinase B (AKT) signaling pathways. BMSCs were extracted from New Zealand white rabbits and used to analyze the effect of glycitin on BMSCs. BMSCs were cleared using xylene and observed via light microscopy. BMSCs were subsequently induced with glycitin (0.01, 0.5, 1, 5 and 10 µM) for 7 days, and stained with Oil Red O. The mechanism of action of glycitin on BMSCs was investigated, in which contact with collagen type I (Col I), alkaline phosphatase (ALP), TGF-β and AKT was studied. Firstly, BMSCs appeared homogeneously mazarine blue, and which showed that BMSCs were successful extracted. Administration of glycitin increased cell proliferation and promoted osteoblast formation from BMSCs. Furthermore, glycitin activated the gene expression of Col I and ALP in BMSCs. Notably, glycitin suppressed protein expression of TGF-β and AKT in BMSCs. These results indicated that glycitin may regulate osteoblasts through TGF-β or AKT signaling pathways in BMSCs. PMID:27882117

  14. [Calcitonin gene-related peptide-induced osteogenic differentiation of mouse bone marrow stromal cells through Hippo pathway in vitro].

    PubMed

    Fei, Wang; Huiyu, Zhang; Yuxin, Dou; Shiting, Li; Gang, Zhang; Yinghui, Tan

    2016-06-01

    Previous studies have clarified that calcitonin gene-related peptide (CGRP) can promote the biologi- cal activity of osteoblasts. To further reveal the role of CGRP in bone repair, we studied its influence on osteogenic differentia- tion of mouse bone marrow stromal cells (BMSCs) and initially explored the effect of the Hippo signaling pathway with this process. BMSCs were induced to osteogenic differentiate osteoblasts by different concentrations of CGRP for a screening of the optimal concentration. CGRP was added in BMSCs, then the activity of alkaline phosphatase (ALP) and the number of mineralized nodules were examined by specific ALP kits after 48 hours and alizarin red staining fluid after 7 days, respectively. The protein expression of p-Mst1/2 was measured by Western blot. Verteporfin was used to block the downstream Yap signaling. The mRNA expression of collagen type I (Col I) and runt-related transcription factor 2 (Runx2) were detected by reverse transcription-polymerase chain reaction. Compared to the blank group, different concentrations of CGRP (10⁻⁹, 10⁻⁸, 10⁻⁷ mol · L⁻¹), especially 10⁻⁸ mol · L⁻¹, significantly increased the ALP activity of BMSCs (P < 0.05). Alizarin red staining also showed more mineralized nodules in 10⁻⁸ mol · L⁻¹ group. The expression of p-Mst1/2 increased in the CGRP group (P < 0.05). Verteporfin treatment effectively decreased the mRNA expression of Runx2 and Col I (P < 0.05). The Hippo signaling pathway plays a role in CGRP-induced osteogenic differentiation in mouse BMSCs.

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

    PubMed Central

    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

  16. Mesenchymal stromal cell derived extracellular vesicles rescue radiation damage to murine marrow hematopoietic cells

    PubMed Central

    Wen, Sicheng; Dooner, Mark; Cheng, Yan; Papa, Elaine; Del Tatto, Michael; Pereira, Mandy; Deng, Yanhui; Goldberg, Laura; Aliotta, Jason; Chatterjee, Devasis; Stewart, Connor; Carpanetto, Andrea; Collino, Federica; Bruno, Stefania; Camussi, Giovanni; Quesenberry, Peter

    2016-01-01

    Mesenchymal stromal cells (MSC) have been shown to reverse radiation damage to marrow stem cells. We have evaluated the capacity of MSC-derived extracellular vesicles (MSC-EVs) to mitigate radiation injury to marrow stem cells at 4 hours to 7 days after irradiation. Significant restoration of marrow stem cell engraftment at 4, 24 and 168 hours post-irradiation by exposure to MSC-EVs was observed at 3 weeks to 9 months after transplant and further confirmed by secondary engraftment. Intravenous injection of MSC-EVs to 500cGy exposed mice led to partial recovery of peripheral blood counts and restoration of the engraftment of marrow. The murine hematopoietic cell line, FDC-P1 exposed to 500 cGy, showed reversal of growth inhibition, DNA damage and apoptosis on exposure to murine or human MSC-EVs. Both murine and human MSC-EVs reverse radiation damage to murine marrow cells and stimulate normal murine marrow stem cell/progenitors to proliferate. A preparation with both exosomes and microvesicles was found to be superior to either microvesicles or exosomes alone. Biologic activity was seen in freshly isolated vesicles and in vesicles stored for up to 6 months in 10% DMSO at −80°C. These studies indicate that MSC-EVs can reverse radiation damage to bone marrow stem cells. PMID:27150009

  17. Role of Nanog in the maintenance of marrow stromal stem cells during post natal bone regeneration

    SciTech Connect

    Bais, Manish V.; Shabin, Zabrina M.; Young, Megan; Einhorn, Thomas A.; Kotton, Darrell N.; Gerstnefeld, Louis C.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Nanog is related to marrow stromal stem cell maintenance. Black-Right-Pointing-Pointer Increasing Nanog expression is seen during post natal surgical bone repair. Black-Right-Pointing-Pointer Nanog knockdown decreases post surgical bone regeneration. -- Abstract: Post natal bone repair elicits a regenerative mechanism that restores the injured tissue to its pre-injury cellular composition and structure and is believed to recapitulate the embryological processes of bone formation. Prior studies showed that Nanog, a central epigenetic regulator associated with the maintenance of embryonic stem cells (ESC) was transiently expressed during fracture healing, Bais et al. . In this study, we show that murine bone marrow stromal cells (MSCs) before they are induced to undergo osteogenic differentiation express {approx}50 Multiplication-Sign the background levels of Nanog seen in murine embryonic fibroblasts (MEFs) and the W20-17 murine marrow stromal cell line stably expresses Nanog at {approx}80 Multiplication-Sign the MEF levels. Nanog expression in this cell line was inhibited by BMP7 treatment and Nanog lentivrial shRNA knockdown induced the expression of the terminal osteogenic gene osteocalcin. Lentivrial shRNA knockdown or lentiviral overexpression of Nanog in bone MSCs had inverse effects on proliferation, with knockdown decreasing and overexpression increasing MSC cell proliferation. Surgical marrow ablation of mouse tibia by medullary reaming led to a {approx}3-fold increase in Nanog that preceded osteogenic differentiation during intramembranous bone formation. Lentiviral shRNA knockdown of Nanog after surgical ablation led to an initial overexpression of osteogenic gene expression with no initial effect on bone formation but during subsequent remodeling of the newly formed bone a {approx}50% decrease was seen in the expression of terminal osteogenic gene expression and a {approx}50% loss in trabecular bone mass. This

  18. Mechanical stimulation promote the osteogenic differentiation of bone marrow stromal cells through epigenetic regulation of Sonic Hedgehog.

    PubMed

    Wang, Chuandong; Shan, Shengzhou; Wang, Chenglong; Wang, Jing; Li, Jiao; Hu, Guoli; Dai, Kerong; Li, Qingfeng; Zhang, Xiaoling

    2017-03-15

    Mechanical unloading leads to bone loss and disuse osteoporosis partly due to impaired osteoblastogenesis of bone marrow stromal cells (BMSCs). However, the underlying molecular mechanisms of this phenomenon are not fully understood. In this study, we demonstrated that cyclic mechanical stretch (CMS) promotes osteoblastogenesis of BMSCs both in vivo and in vitro. Besides, we found that Hedgehog (Hh) signaling pathway was activated in this process. Inhibition of which by either knockdown of Sonic hedgehog (Shh) or treating BMSCs with Hh inhibitors attenuated the osteogenic effect of CMS on BMSCs, suggesting that Hh signaling pathway acts as an endogenous mediator of mechanical stimuli on BMSCs. Furthermore, we demonstrated that Shh expression level was regulated by DNA methylation mechanism. Chromatin Immunoprecipitation (ChIP) assay showed that DNA methyltransferase 3b (Dnmt3b) binds to Shh gene promoter, leading to DNA hypermethylation in mechanical unloading BMSCs. However, mechanical stimulation down-regulates the protein level of Dnmt3b, results in DNA demethylation and Shh expression. More importantly, we found that inhibition of Dnmt3b partly rescued bone loss in HU mice by mechanical unloading. Our results demonstrate, for the first time, that mechanical stimulation regulates osteoblastic genes expression via direct regulation of Dnmt3b, and the therapeutic inhibition of Dnmt3b may be an efficient strategy for enhancing bone formation under mechanical unloading. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    PubMed

    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.

  20. Flow perfusion enhances the calcified matrix deposition of marrow stromal cells in biodegradable nonwoven fiber mesh scaffolds.

    PubMed

    Sikavitsas, Vassilios I; Bancroft, Gregory N; Lemoine, Jeremy J; Liebschner, Michael A K; Dauner, Martin; Mikos, Antonios G

    2005-01-01

    In this study, we report on the ability of resorbable poly(L-lactic acid) (PLLA) nonwoven scaffolds to support the attachment, growth, and differentiation of marrow stromal cells (MSCs) under fluid flow. Rat MSCs were isolated from young male Wistar rats and expanded using established methods. The cells were then seeded on PLLA nonwoven fiber meshes. The PLLA nonwoven fiber meshes had 99% porosity, 17 microm fiber diameter, 10 mm scaffold diameter, and 1.7-mm thickness. The nonwoven PLLA meshes were seeded with a cell suspension of 5 x 10(5) cells in 300 microl, and cultured in a flow perfusion bioreactor and under static conditions. Cell/polymer nonwoven scaffolds cultured under flow perfusion had significantly higher amounts of calcified matrix deposited on them after 16 days of culture. Microcomputed tomography revealed that the in vitro generated extracellular matrix in the scaffolds cultured under static conditions was denser at the periphery of the scaffold while in the scaffolds cultured in the perfusion bioreactor the extracellular matrix demonstrated a more homogeneous distribution. These results show that flow perfusion accelerates the proliferation and differentiation of MSCs, seeded on nonwoven PLLA scaffolds, toward the osteoblastic phenotype, and improves the distribution of the in vitro generated calcified extracellular matrix.

  1. Selection of highly osteogenic and chondrogenic cells from bone marrow stromal cells in biocompatible polymer-coated plates.

    PubMed

    Liu, G; Iwata, K; Ogasawara, T; Watanabe, J; Fukazawa, K; Ishihara, K; Asawa, Y; Fujihara, Y; Chung, U-L; Moro, T; Takatori, Y; Takato, T; Nakamura, K; Kawaguchi, H; Hoshi, K

    2010-03-15

    To enrich the subpopulation that preserves self-renewal and multipotentiality from conventionally prepared bone marrow stromal cells (MSCs), we attempted to use 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer-coated plates that selected the MSCs with strong adhesion ability and evaluated the proliferation ability or osteogenic/chondrogenic potential of the MPC polymer-selected MSCs. The number of MSCs that were attached to the MPC polymer-coated plates decreased with an increase in the density of MPC unit (0-10%), whereas no significant difference in the proliferation ability was seen among these cells. The surface epitopes of CD29, CD44, CD105, and CD166, and not CD34 or CD45, were detectable in the cells of all MPC polymer-coated plates, implying that they belong to the MSC category. In the osteogenic and chondrogenic induction, the MSCs selected by the 2-5% MPC unit composition showed higher expression levels of osteoblastic and chondrocytic markers (COL1A1/ALP, or COL2A1/COL10A1/Sox9) at passage 2, compared with those of 0-1% or even 10% MPC unit composition, while the enhanced effects continued by passage 5. The selection based on the adequate cell adhesiveness by the MPC polymer-coated plates could improve the osteogenic and chondrogenic potential of MSCs, which would provide cell sources that can be used to treat the more severe and various bone/cartilage diseases.

  2. The influence of stereolithographic scaffold architecture and composition on osteogenic signal expression with rat bone marrow stromal cells

    PubMed Central

    Kim, Kyobum; Dean, David; Wallace, Jonathan; Breithaupt, Rob; Mikos, Antonios G.; Fisher, John P.

    2011-01-01

    Scaffold design parameters, especially physical construction factors such as mechanical stiffness of substrate materials, pore size of 3D porous scaffolds, and channel geometry, are known to influence the osteogenic signal expression and subsequent differentiation of a transplanted cell population. In this study of photocrosslinked poly(propylene fumarate) (PPF) and diethyl fumarate (DEF) scaffolds, the effect of DEF incorporation ratio and pore size on the osteogenic signal expression of rat bone marrow stromal cells (BMSCs) was investigated. Results demonstrated that DEF concentrations and pore sizes that led to increased scaffold mechanical stiffness also upregulated osteogenic signal expression, including bone morphogenic protein-2 (BMP-2), fibroblast growth factors-2 (FGF-2), transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and Runx2 transcriptional factor. Similar scaffold fabrication parameters supported rapid BMSC osteoblastic differentiation, as demonstrated by increased alkaline phosphatase (ALP) and osteocalcin expression. When scaffolds with random architecture, fabricated by porogen leaching, were compared to those with controlled architecture, fabricated by stereolithography (SLA), results showed that SLA scaffolds with the highly permeable and porous channels also have significantly higher expression of FGF-2, TGF-β1, and VEGF. Subsequent ALP expression and osteopontin secretion were also significantly increased in SLA scaffolds. Based upon these results, we conclude that scaffold properties provided by additive manufacturing techniques such as SLA fabrication, particularly increased mechanical stiffness and high permeability, may stimulate dramatic BMSC responses that promote rapid bone tissue regeneration. PMID:21396709

  3. The Role of the Bone Marrow Stromal Compartment in the Hematopoietic Response to Microbial Infections

    PubMed Central

    Nombela-Arrieta, César; Isringhausen, Stephan

    2017-01-01

    Continuous production of blood cells unfolds within a complex three-dimensional tissue scaffold established by highly organized stromal cell networks of mesenchymal, neural, and vascular origin inside bone marrow (BM) cavities. Collectively, stromal cells have been shown to serve two principal roles; first as primary participants of bone remodeling and metabolism and second as master regulators of different stages of blood cell development and production. Indeed, ample evidence demonstrates that stromal cells can sense and integrate systemic signals to shape hematopoietic responses and that these regulatory mechanisms are subverted in multiple pathologic conditions. Microbial infections are stressors that elicit potent inflammatory reactions and induce substantial alterations of hematopoietic output. Whether the cellular components of the BM stromal microenvironment are targeted by infections and participate in infection-induced hematopoiesis has not been investigated in sufficient detail to date. In this manuscript, we provide a succinct updated overview of the different cell populations that are currently known to form BM stroma. We discuss experimental evidence demonstrating that different stromal components are actively damaged or functionally altered by pathogens and/or ensuing inflammatory signals and review how these effects are known to contribute to the hematologic manifestations observed during infections. PMID:28163704

  4. Ephrin B1 regulates bone marrow stromal cell differentiation and bone formation by influencing TAZ transactivation via complex formation with NHERF1.

    PubMed

    Xing, Weirong; Kim, Jonghyun; Wergedal, Jon; Chen, Shin-Tai; Mohan, Subburaman

    2010-02-01

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

  5. Gene expression profiling of bone marrow mesenchymal stem cells from Osteogenesis Imperfecta patients during osteoblast differentiation.

    PubMed

    Kaneto, Carla Martins; Pereira Lima, Patrícia S; Prata, Karen Lima; Dos Santos, Jane Lima; de Pina Neto, João Monteiro; Panepucci, Rodrigo Alexandre; Noushmehr, Houtan; Covas, Dimas Tadeu; de Paula, Francisco José Alburquerque; Silva, Wilson Araújo

    2017-06-01

    Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differentiate into osteoblasts, adipocytes and chondroblasts that have gained great importance as a source for cell therapy. Recently, a number of studies involving the analysis of gene expression of undifferentiated MSCs and of MSCs in the differentiation into multiple lineage processes were observed but there is no information concerning the gene expression of MSCs from Osteogenesis Imperfecta (OI) patients. Osteogenesis Imperfecta is characterized as a genetic disorder in which a generalized osteopenia leads to excessive bone fragility and severe bone deformities. The aim of this study was to analyze gene expression profile during osteogenic differentiation from BMMSCs (Bone Marrow Mesenchymal Stem Cells) obtained from patients with Osteogenesis Imperfecta and from control subjects. Bone marrow samples were collected from three normal subjects and five patients with OI. Mononuclear cells were isolated for obtaining mesenchymal cells that had been expanded until osteogenic differentiation was induced. RNA was harvested at seven time points during the osteogenic differentiation period (D0, D+1, D+2, D+7, D+12, D+17 and D+21). Gene expression analysis was performed by the microarray technique and identified several differentially expressed genes. Some important genes for osteoblast differentiation had lower expression in OI patients, suggesting a smaller commitment of these patient's MSCs with the osteogenic lineage. Other genes also had their differential expression confirmed by RT-qPCR. An increase in the expression of genes related to adipocytes was observed, suggesting an increase of adipogenic differentiation at the expense osteogenic differentiation. Copyright © 2017. Published by Elsevier Masson SAS.

  6. Gene expression profile of human bone marrow stromal cells: high-throughput expressed sequence tag sequencing analysis.

    PubMed

    Jia, Libin; Young, Marian F; Powell, John; Yang, Liming; Ho, Nicola C; Hotchkiss, Robert; Robey, Pamela Gehron; Francomano, Clair A

    2002-01-01

    Human bone marrow stromal cells (HBMSC) are pluripotent cells with the potential to differentiate into osteoblasts, chondrocytes, myelosupportive stroma, and marrow adipocytes. We used high-throughput DNA sequencing analysis to generate 4258 single-pass sequencing reactions (known as expressed sequence tags, or ESTs) obtained from the 5' (97) and 3' (4161) ends of human cDNA clones from a HBMSC cDNA library. Our goal was to obtain tag sequences from the maximum number of possible genes and to deposit them in the publicly accessible database for ESTs (dbEST of the National Center for Biotechnology Information). Comparisons of our EST sequencing data with nonredundant human mRNA and protein databases showed that the ESTs represent 1860 gene clusters. The EST sequencing data analysis showed 60 novel genes found only in this cDNA library after BLAST analysis against 3.0 million ESTs in NCBI's dbEST database. The BLAST search also showed the identified ESTs that have close homology to known genes, which suggests that these may be newly recognized members of known gene families. The gene expression profile of this cell type is revealed by analyzing both the frequency with which a message is encountered and the functional categorization of expressed sequences. Comparing an EST sequence with the human genomic sequence database enables assignment of an EST to a specific chromosomal region (a process called digital gene localization) and often enables immediate partial determination of intron/exon boundaries within the genomic structure. It is expected that high-throughput EST sequencing and data mining analysis will greatly promote our understanding of gene expression in these cells and of growth and development of the skeleton.

  7. Transcriptomic profile induced in bone marrow mesenchymal stromal cells after interaction with multiple myeloma cells: implications in myeloma progression and myeloma bone disease

    PubMed Central

    Garcia-Gomez, Antonio; Las Rivas, Javier De; Ocio, Enrique M.; Díaz-Rodríguez, Elena; Montero, Juan C.; Martín, Montserrat; Blanco, Juan F.; Sanchez-Guijo, Fermín M.; Pandiella, Atanasio; San Miguel, Jesús F.; Garayoa, Mercedes

    2014-01-01

    Despite evidence about the implication of the bone marrow (BM) stromal microenvironment in multiple myeloma (MM) cell growth and survival, little is known about the effects of myelomatous cells on BM stromal cells. Mesenchymal stromal cells (MSCs) from healthy donors (dMSCs) or myeloma patients (pMSCs) were co-cultured with the myeloma cell line MM.1S, and the transcriptomic profile of MSCs induced by this interaction was analyzed. Deregulated genes after co-culture common to both d/pMSCs revealed functional involvement in tumor microenvironment cross-talk, myeloma growth induction and drug resistance, angiogenesis and signals for osteoclast activation and osteoblast inhibition. Additional genes induced by co-culture were exclusively deregulated in pMSCs and predominantly associated to RNA processing, the ubiquitine-proteasome pathway, cell cycle regulation, cellular stress and non-canonical Wnt signaling. The upregulated expression of five genes after co-culture (CXCL1, CXCL5 and CXCL6 in d/pMSCs, and Neuregulin 3 and Norrie disease protein exclusively in pMSCs) was confirmed, and functional in vitro assays revealed putative roles in MM pathophysiology. The transcriptomic profile of pMSCs co-cultured with myeloma cells may better reflect that of MSCs in the BM of myeloma patients, and provides new molecular insights to the contribution of these cells to MM pathophysiology and to myeloma bone disease. PMID:25268740

  8. Transcriptomic profile induced in bone marrow mesenchymal stromal cells after interaction with multiple myeloma cells: implications in myeloma progression and myeloma bone disease.

    PubMed

    Garcia-Gomez, Antonio; De Las Rivas, Javier; Ocio, Enrique M; Díaz-Rodríguez, Elena; Montero, Juan C; Martín, Montserrat; Blanco, Juan F; Sanchez-Guijo, Fermín M; Pandiella, Atanasio; San Miguel, Jesús F; Garayoa, Mercedes

    2014-09-30

    Despite evidence about the implication of the bone marrow (BM) stromal microenvironment in multiple myeloma (MM) cell growth and survival, little is known about the effects of myelomatous cells on BM stromal cells. Mesenchymal stromal cells (MSCs) from healthy donors (dMSCs) or myeloma patients (pMSCs) were co-cultured with the myeloma cell line MM.1S, and the transcriptomic profile of MSCs induced by this interaction was analyzed. Deregulated genes after co-culture common to both d/pMSCs revealed functional involvement in tumor microenvironment cross-talk, myeloma growth induction and drug resistance, angiogenesis and signals for osteoclast activation and osteoblast inhibition. Additional genes induced by co-culture were exclusively deregulated in pMSCs and predominantly associated to RNA processing, the ubiquitine-proteasome pathway, cell cycle regulation, cellular stress and non-canonical Wnt signaling. The upregulated expression of five genes after co-culture (CXCL1, CXCL5 and CXCL6 in d/pMSCs, and Neuregulin 3 and Norrie disease protein exclusively in pMSCs) was confirmed, and functional in vitro assays revealed putative roles in MM pathophysiology. The transcriptomic profile of pMSCs co-cultured with myeloma cells may better reflect that of MSCs in the BM of myeloma patients, and provides new molecular insights to the contribution of these cells to MM pathophysiology and to myeloma bone disease.

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1998-01-01

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

  11. Bone deficit in ovariectomized rats. Functional contribution of the marrow stromal cell population and the effect of oral dihydrotachysterol treatment.

    PubMed Central

    Tabuchi, C; Simmons, D J; Fausto, A; Russell, J E; Binderman, I; Avioli, L V

    1986-01-01

    This study investigates the proliferative and osteogenic role of marrow stromal/osteoprogenitor cells in the development of the cortical bone deficit in ovariectomized (OVX) female rats. In vitro, clonal growth of marrow stromal cells from OVX rats was significantly impaired (vs. sham-operated controls). Yet in vivo, cells from sham-operated and OVX rats had equal osteogenic potential in several in vivo experimental situations, such as in intraperitoneally implanted millipore diffusion chambers and in intramuscular implants of marrow plus osteoinductive bone matrix (composite grafts). Long-term (6 mo) dihydrotachysterol (DHT) treatment of OVX rats enhanced their in vitro proliferative potential and clonal growth, as well as their osteogenic expression in composite grafts. The observation that the in vivo osteogenic performance of OVX rat marrow stromal cells was normal at extraosseous sites suggests that the mechanisms leading to osteopenia may involve an abnormality in cell-matrix interactions. PMID:3528218

  12. Connective tissue growth factor is expressed in bone marrow stromal cells and promotes interleukin-7-dependent B lymphopoiesis

    PubMed Central

    Cheung, Laurence C.; Strickland, Deborah H.; Howlett, Meegan; Ford, Jette; Charles, Adrian K.; Lyons, Karen M.; Brigstock, David R.; Goldschmeding, Roel; Cole, Catherine H.; Alexander, Warren S.; Kees, Ursula R.

    2014-01-01

    Hematopoiesis occurs in a complex bone marrow microenvironment in which bone marrow stromal cells provide critical support to the process through direct cell contact and indirectly through the secretion of cytokines and growth factors. We report that connective tissue growth factor (Ctgf, also known as Ccn2) is highly expressed in murine bone marrow stromal cells. In contrast, connective tissue growth factor is barely detectable in unfractionated adult bone marrow cells. While connective tissue growth factor has been implicated in hematopoietic malignancies, and is known to play critical roles in skeletogenesis and regulation of bone marrow stromal cells, its role in hematopoiesis has not been described. Here we demonstrate that the absence of connective tissue growth factor in mice results in impaired hematopoiesis. Using a chimeric fetal liver transplantation model, we show that absence of connective tissue growth factor has an impact on B-cell development, in particular from pro-B to more mature stages, which is linked to a requirement for connective tissue growth factor in bone marrow stromal cells. Using in vitro culture systems, we demonstrate that connective tissue growth factor potentiates B-cell proliferation and promotes pro-B to pre-B differentiation in the presence of interleukin-7. This study provides a better understanding of the functions of connective tissue growth factor within the bone marrow, showing the dual regulatory role of the growth factor in skeletogenesis and in stage-specific B lymphopoiesis. PMID:24727816

  13. The differentiation directions of the bone marrow stromal cells under modeling microgravity

    NASA Astrophysics Data System (ADS)

    Nesterenko, Olga; Rodionova, Natalia; Katkova, Olena

    Within experiments on rats simulating microgravity by base load remove from back limbs (duration of the experiment 1,5 months) on marrow stromal cells cultures (ex vivo, in vitro) comprising osteogenic cells-predecessors, extracted from femurs, studied their peculiarities of the colony formation ablity, the cell structure, some cytological and ultra-structural characteristics and differentiation direction. It was found that that under microgravity conditions there is a decline of the stromal cells colony formation intensity, decrease of the colonies size and cells mitotic activity that indicates decrease of their growth potential. Both in control and in experiment the colonies were presented by population of low-differentiated cells, differentiated cells and mature cells. The comparative cytological and morphometric analysis have shown that the studied stromal cells in colonies have the smaller sizes, more elongated shape, and higher nucleocytoplasmic ratio. Cells composition in the experiment colonies is reliably different by the ratio of the low-differentiating to being differentiated cells; a ratio of low-differentiated to already differentiated cells; ratio of differentiated cells to total number of all cells. In comparison with control group, amount of the cells passed trough a differentiation stage and mature cells in colonies is decreased by 3 to 4 times. Among the differentiated stromal cells in colonies increasing amount of adipocytes was revealed. The analysis of electron microscope microphotographs showed that in osteogenic cells differentiated under microgravity conditions, there is a reduction of the specific volume of a granular endoplasmic reticulum, Golgi's complex and quantity of nuclei reduction that indicates depression of the specific biosyntheses process intensity in cells. The increase of lysosomes and myelinic structures quantity is linked to organelles partial reduction. Consolidation of mitochondrias is an evidence of the cells’ energy

  14. Bone marrow derived macrophages fuse with intestine stromal cells and contribute to chronic fibrosis after radiation.

    PubMed

    Yeh, Ming-Han; Chang, Ya-Hui; Tsai, Yi-Chih; Chen, Su-Liang; Huang, Tze-Sing; Chiu, Jeng-Fong; Ch'ang, Hui-Ju

    2016-05-01

    Bone marrow-derived cells (BMDC) have been demonstrated to play a critical role in intestine regeneration. However, organ fibrosis was one of the major side effects of bone marrow (BM) transplantation. It warrants further investigation on the mechanisms of BM cell therapy in radiation induced intestine damage. We established three murine models to evaluate BMDC within intestines after radiation, including cre-loxP system of transgenic mice. In vitro co-culture between murine BM with human intestine stromal cells was also performed to measure the level of fusion and fibrosis after treatment with anti-fibrotic agents or after macrophage depletion. Despite complete recovery of epithelial mucosa from radiation damage, we found persistent proliferation and repopulation of BMDC within the lamina propria. Fusion between BM derived monocytic and intestine stromal cells correlated with the level of fibrosis and proliferation index. Depleting macrophages genetically using CD11b-DTR mouse model or pharmacologically using clodronate liposome reduced the level of cell fusion and intestine fibrosis. Fibrotic cues from intestine enhance fusion between BM-derived monocytes/macrophages with intestine stromal cells. The fusion hybrids promote cell cycle re-entry, proliferation and reinforce fibrosis signal. Depleting macrophages interferes with cell fusion and ameliorates radiation-induced intestine fibrosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. The use of bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for alveolar bone tissue engineering: basic science to clinical translation.

    PubMed

    Kagami, Hideaki; Agata, Hideki; Inoue, Minoru; Asahina, Izumi; Tojo, Arinobu; Yamashita, Naohide; Imai, Kohzoh

    2014-06-01

    Bone tissue engineering is a promising field of regenerative medicine in which cultured cells, scaffolds, and osteogenic inductive signals are used to regenerate bone. Human bone marrow stromal cells (BMSCs) are the most commonly used cell source for bone tissue engineering. Although it is known that cell culture and induction protocols significantly affect the in vivo bone forming ability of BMSCs, the responsible factors of clinical outcome are poorly understood. The results from recent studies using human BMSCs have shown that factors such as passage number and length of osteogenic induction significantly affect ectopic bone formation, although such differences hardly affected the alkaline phosphatase activity or gene expression of osteogenic markers. Application of basic fibroblast growth factor helped to maintain the in vivo osteogenic ability of BMSCs. Importantly, responsiveness of those factors should be tested under clinical circumstances to improve the bone tissue engineering further. In this review, clinical application of bone tissue engineering was reviewed with putative underlying mechanisms.

  16. The role of osteoblasts in regulating hematopoietic stem cell activity and tumor metastasis.

    PubMed

    Neiva, K; Sun, Y-X; Taichman, R S

    2005-10-01

    Bone marrow stromal cells are critical regulators of hematopoiesis. Osteoblasts are part of the stromal cell support system in bone marrow and may be derived from a common precursor. Several studies suggested that osteoblasts regulate hematopoiesis, yet the entire mechanism is not understood. It is clear, however, that both hematopoietic precursors and osteoblasts interact for the production of osteoclasts and the activation of resorption. We observed that hematopoietic stem cells (HSCs) regulate osteoblastic secretion of various growth factors, and that osteoblasts express some soluble factors exclusively in the presence of HSCs. Osteoblasts and hematopoietic cells are closely associated with each other in the bone marrow, suggesting a reciprocal relationship between them to develop the HSC niche. One critical component regulating the niche is stromal-derived factor-1 (SDF-1) and its receptor CXCR4 which regulates stem cell homing and, as we have recently demonstrated, plays a crucial role in facilitating those tumors which metastasize to bone. Osteoblasts produce abundant amounts of SDF-1 and therefore osteoblasts play an important role in metastasis. These findings are discussed in the context of the role of osteoblasts in marrow function in health and disease.

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

    SciTech Connect

    Perkins, S.; Fleischman, R.A.

    1988-04-01

    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.

  18. Prostaglandin E2 acts via bone marrow macrophages to block PTH-stimulated osteoblast differentiation in vitro.

    PubMed

    Choudhary, Shilpa; Blackwell, Katherine; Voznesensky, Olga; Deb Roy, Abhijit; Pilbeam, Carol

    2013-09-01

    Intermittent PTH is the major anabolic therapy for osteoporosis while continuous PTH causes bone loss. PTH acts on the osteoblast (OB) lineage to regulate bone resorption and formation. PTH also induces cyclooxygenase-2 (COX-2), producing prostaglandin E2 (PGE(2)) that can act on both OBs and osteoclasts (OCs). Because intermittent PTH is more anabolic in Cox-2 knockout (KO) than wild type (WT) mice, we hypothesized COX-2 might contribute to the effects of continuous PTH by suppressing PTH-stimulated differentiation of mesenchymal stem cells into OBs. We compared effects of continuous PTH on bone marrow stromal cells (BMSCs) and primary OBs (POBs) from Cox-2 KO mice, mice with deletion of PGE(2) receptors (Ptger(4) and Ptger(2) KO mice), and WT controls. PTH increased OB differentiation in BMSCs only in the absence of COX-2 expression or activity. In the absence of COX-2, PTH stimulated differentiation if added during the first week of culture. In Cox-2 KO BMSCs, PTH-stimulated differentiation was prevented by adding PGE(2) to cultures. Co-culture of POBs with M-CSF-expanded bone marrow macrophages (BMMs) showed that the inhibition of PTH-stimulated OB differentiation required not only COX-2 or PGE(2) but also BMMs. Sufficient PGE(2) to mediate the inhibitory effect was made by either WT POBs or WT BMMs. The inhibitory effect mediated by COX-2/PGE(2) was transferred by conditioned media from RANKL-treated BMMs and could be blocked by osteoprotegerin, which interferes with RANKL binding to its receptor on OC lineage cells. Deletion of Ptger(4), but not Ptger(2), in BMMs prevented the inhibition of PTH-stimulated OB differentiation. As expected, PGE(2) also stimulated OB differentiation, but when given in combination with PTH, the stimulatory effects of both were abrogated. These data suggest that PGE(2), acting via EP4R on BMMs committed to the OC lineage, stimulated secretion of a factor or factors that acted to suppress PTH-stimulated OB differentiation. This

  19. Prostaglandin E2 acts via bone marrow macrophages to block PTH-stimulated osteoblast differentiation in vitro

    PubMed Central

    Choudhary, Shilpa; Blackwell, Katherine; Voznesensky, Olga; Roy, Abhijit Deb; Pilbeam, Carol

    2014-01-01

    Intermittent PTH is the major anabolic therapy for osteoporosis while continuous PTH causes bone loss. PTH acts on the osteoblast (OB) lineage to regulate bone resorption and formation. PTH also induces cyclooxygenase-2 (COX-2), producing prostaglandin E2 (PGE2) that can act on both OBs and osteoclasts (OCs). Because intermittent PTH is more anabolic in Cox-2 knockout (KO) than wild type (WT) mice, we hypothesized COX-2 might contribute to the effects of continuous PTH by suppressing PTH-stimulated differentiation of mesenchymal stem cells into OBs. We compared effects of continuous PTH on bone marrow stromal cells (BMSCs) and primary OBs (POBs) from Cox-2 KO mice, mice with deletion of PGE2 receptors (Ptger4 and Ptger2 KO mice), and WT controls. PTH increased OB differentiation in BMSCs only in the absence of COX-2 expression or activity. In the absence of COX-2, PTH stimulated differentiation if added during the first week of culture. In Cox-2 KO BMSCs, PTH-stimulated differentiation was prevented by adding PGE2 to cultures. Co-culture of POBs with M-CSF-expanded bone marrow macrophages (BMMs) showed that the inhibition of PTH-stimulated OB differentiation required not only COX-2 or PGE2 but also BMMs. Sufficient PGE2 to mediate the inhibitory effect was made by either WT POBs or WT BMMs. The inhibitory effect mediated by COX-2/PGE2 was transferred by conditioned media from RANKL-treated BMMs and could be blocked by osteoprotegerin, which interferes with RANKL binding to its receptor on OC lineage cells. Deletion of Ptger4, but not Ptger2, in BMMs prevented the inhibition of PTH-stimulated OB differentiation. As expected, PGE2 also stimulated OB differentiation, but when given in combination with PTH, the stimulatory effects of both were abrogated. These data suggest that PGE2, acting via EP4R on BMMs committed to the OC lineage, stimulated secretion of a factor or factors that acted to suppress PTH-stimulated OB differentiation. This suppression of OB

  20. Nanostructured TiO2 Surfaces Promote Human Bone Marrow Mesenchymal Stem Cells Differentiation to Osteoblasts

    PubMed Central

    Vercellino, Marco; Ceccarelli, Gabriele; Cristofaro, Francesco; Balli, Martina; Bertoglio, Federico; Bruni, Gianna; Benedetti, Laura; Avanzini, Maria Antonietta; Imbriani, Marcello; Visai, Livia

    2016-01-01

    Micro- and nano-patterning/modification are emerging strategies to improve surfaces properties that may influence critically cells adherence and differentiation. Aim of this work was to study the in vitro biological reactivity of human bone marrow mesenchymal stem cells (hBMSCs) to a nanostructured titanium dioxide (TiO2) surface in comparison to a coverglass (Glass) in two different culture conditions: with (osteogenic medium (OM)) and without (proliferative medium (PM)) osteogenic factors. To evaluate cell adhesion, hBMSCs phosphorylated focal adhesion kinase (pFAK) foci were analyzed by confocal laser scanning microscopy (CLSM) at 24 h: the TiO2 surface showed a higher number of pFAK foci with respect to Glass. The hBMSCs differentiation to osteoblasts was evaluated in both PM and OM culture conditions by enzyme-linked immunosorbent assay (ELISA), CLSM and real-time quantitative reverse transcription PCR (qRT-PCR) at 28 days. In comparison with Glass, TiO2 surface in combination with OM conditions increased the content of extracellular bone proteins, calcium deposition and alkaline phosphatase activity. The qRT-PCR analysis revealed, both in PM and OM, that TiO2 surface increased at seven and 28 days the expression of osteogenic genes. All together, these results demonstrate the capability of TiO2 nanostructured surface to promote hBMSCs osteoblast differentiation and its potentiality in biomedical applications. PMID:28335251

  1. Characterization of Insulin-Secreting Porcine Bone Marrow Stromal Cells Ex Vivo and Autologous Cell Therapy In Vivo.

    PubMed

    Do, Hai Van Thi; Loke, Wan Ting; Kee, Irene; Liang, Vivienne; David, Sebastian J; Gan, Shu Uin; Lee, Sze Sing; Ng, Wai Har; Koong, Heng Nung; Ong, Hock Soo; Lee, Kok Onn; Calne, Roy Y; Kon, Oi Lian

    2015-01-01

    Cell therapy could potentially meet the need for pancreas and islet transplantations in diabetes mellitus that far exceeds the number of available donors. Bone marrow stromal cells are widely used in clinical trials mainly for their immunomodulatory effects with a record of safety. However, less focus has been paid to developing these cells for insulin secretion by transfection. Although murine models of diabetes have been extensively used in gene and cell therapy research, few studies have shown efficacy in large preclinical animal models. Here we report optimized conditions for ex vivo expansion and characterization of porcine bone marrow stromal cells and their permissive expression of a transfected insulin gene. Our data show that these cells resemble human bone marrow stromal cells in surface antigen expression, are homogeneous, and can be reproducibly isolated from outbred Yorkshire-Landrace pigs. Porcine bone marrow stromal cells were efficiently expanded in vitro to >10(10) cells from 20 ml of bone marrow and remained karyotypically normal during expansion. These cells were electroporated with an insulin expression plasmid vector with high efficiency and viability, and secreted human insulin and C-peptide indicating appropriate processing of proinsulin. We showed that autologous insulin-secreting bone marrow stromal cells implanted and engrafted in the liver of a streptozotocin-diabetic pig that modeled type 1 diabetes resulted in partial, but significant, improvement in hyperglycemia that could not be ascribed to regeneration of endogenous β-cells. Glucose-stimulated insulin secretion in vivo from implanted cells in the treated pig was documented by a rise in serum human C-peptide levels during intravenous glucose tolerance tests. Compared to a sham-treated control pig, this resulted in significantly reduced fasting hyperglycemia, a slower rise in serum fructosamine, and prevented weight loss. Taken together, this study suggests that bone marrow stromal

  2. Therapeutic benefit of intrathecal injection of marrow stromal cells on ischemia-injured spinal cord.

    PubMed

    Shi, Enyi; Kazui, Teruhisa; Jiang, Xiaojing; Washiyama, Naoki; Yamashita, Katsushi; Terada, Hitoshi; Bashar, Abul Hasan Muhammad

    2007-04-01

    Prophylactic transplantation of marrow stromal cells (MSCs) before spinal cord ischemia has been shown to attenuate neurologic injures. We sought to investigate the therapeutic effect of MSCs on ischemia-injured spinal cord. Marrow stromal cells were expanded in vitro and prelabeled with bromodeoxyuridine. Spinal cord ischemia was induced in rabbits by infrarenal aortic occlusion for 30 minutes. Four groups were enrolled. About 1 x 10(8) MSCs were intrathecally injected 2 hours (group MSC-2h), 24 hours (group MSC-24h), or 48 hours (group MSC-48h) after spinal cord ischemia, respectively. The control group received intrathecal injection of medium alone. Hind-limb motor function was assessed during a 28-day recovery period with Tarlov criteria, and then histologic examination was performed. Marrow stromal cells still could be found in the spinal cord 4 weeks after transplantation. The capillary density in the ventral gray matter was significantly increased in the three MSC-treated groups (p < 0.01 versus control group, respectively). After a 28-day recovery, marked functional improvement was detected in group MSC-2h (from day 1 to 28, p < 0.05, versus control group, respectively) and group MSC-24h (from day 14 to 28, p < 0.05, versus control group, respectively), but not in group MSC-48h. The number of intact motor neurons was much greater in group MSC-2h (p < 0.05, versus control group). Intrathecal injection of MSCs enhances angiogenesis in the host spinal cord and improves the motor functional recovery after spinal cord ischemia. The therapeutic time window is critical for the therapeutic effect of MSCs.

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

    PubMed Central

    Mindaye, Samuel T.; Lo Surdo, Jessica; Bauer, Steven R.; Alterman, Michail A.

    2015-01-01

    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. PMID:26702413

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

    SciTech Connect

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

    2008-08-08

    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.

  5. β-Caryophyllene promotes osteoblastic mineralization, and suppresses osteoclastogenesis and adipogenesis in mouse bone marrow cultures in vitro

    PubMed Central

    Yamaguchi, Masayoshi; Levy, Robert M.

    2016-01-01

    Osteoporosis is induced by the reduction in bone mass through decreased osteoblastic osteogenesis and increased osteoclastic bone resorption, and it is associated with obesity and diabetes. Osteoblasts and adipocytes are derived from bone marrow mesenchymal stem cells. The prevention of osteoporosis is an important public health concern in aging populations. β-caryophyllene, a component of various essential oils, is a selective agonist of the cannabinoid receptor type 2 and exerts cannabimimetic anti-inflammatory effects in animals. The present study aimed to identify the effect of β-caryophyllene on adipogenesis, osteoblastic mineralization and osteoclastogenesis in mouse bone marrow cell cultures in vitro. Bone marrow cells obtained from mouse femoral tissues were cultured in the presence of β-caryophyllene (0.1–100 µM) in vitro. The results revealed that β-caryophyllene stimulated osteoblastic mineralization, and suppressed adipogenesis and osteoclastogenesis. Thus, β-caryophyllene may be used as a therapeutic agent for the prevention and treatment of osteoporosis. PMID:28105093

  6. [Effect of extracellular matrix components on adhesion of bone marrow multipotent mesenchymal stromal cells to polytetrafluoroethylene].

    PubMed

    Karpenko, A A; Rozanova, I A; Poveshchenko, O V; Lykov, A P; Bondarenko, N A; Kim, I I; Nikonorova, Iu V; Podkhvatilina, N A; Sergeevichev, D S; Popova, I V; Konenkov, V I

    2015-01-01

    Search for new bioengineering materials for creation of small-diameter vascular grafts is currently a priority task. One of the promising trends of creating tissue engineering constructions is coating the internal layer of implants made of polytetrafluoroethylene (PTFE) with autologous mesenchymal multipotent stromal cells. In the study we assessed the ability of separate components of the extracellular matrix such as fibronectin, type I collagen and type IV collagen to influence adhesion, proliferation and morphology of mesenchymal multipotent stromal cells being cultured on PTFE. Bone marrow multipotent stromal cells taken from second-passage Wistar rats in the amount of 106 per 1 cm2 were applied onto PTFE. We used the following variants of preliminary treatment of the material prior to seeding: fibronectin with type I collagen, fibronectin with type IV collagen, fibronectin with a mixture of type I and IV collagens, as well as a control group without coating. After six weeks of cell growth on PTFE patches the samples were subjected to fixation in 10% formalin followed by haematoxylin-eosin stain and morphometric assessment of adhered cells by calculation using the software AxioVision (Carl Zeiss), assessing the number of cells, area of the cellular monolayer, dimensions and ratios of the area of separate cells and the area of cellular nuclei. The maximal area of the monolayer from mesenchymal multipotent stromal cells on the PTFE surface was revealed while culturing with a mixture of fibronectin and type I and IV collagens. Cell colonization density while treatment of the synthetic material with mixtures of fibronectin with type I collagen, type IV collagen and type I and IV collagens demonstrated the results exceeding the parameters of the control specimen 5-, 2.5- and 7-fold, respectively. Hence, extracellular matrix components considerably increase enhance adhesion of cells to PTFE, as well as improve formation of a monolayer from mesenchymal multipotent

  7. The Influence of Aspiration Volume on the Number of Osteoblastic Progenitors Obtained From Bone Marrow in Children.

    PubMed

    Yandow, Suzanne M; Van de Velde, Samuel K; Siebert, Jeanne; Perkins, Sherrie L

    2017-01-30

    Autologous bone marrow aspirates are utilized to treat various conditions in children. The biological value of bone marrow aspirate depends on the concentration of competent osteoblastic progenitors present in the aspirate. It has been shown in adults that increasing bone marrow aspiration volume beyond 2 mL decreases the concentration of osteoblast progenitor cells because of dilution of the sample with peripheral blood. The effect of varying bone marrow aspiration volumes on the osteoblast cell content has not been determined in children. In total, 21 children (3 male and 18 female patients, age range 8 mo to 14 y) scheduled for pelvic osteotomy were included in the study. Three separate bone marrow aspirates of 1, 5, and 10 mL were obtained from the anterior superior iliac crest. Total number of nucleated cells was counted per aspirate and the prevalence of alkaline phosphatase-positive colony-forming units was determined per million nucleated cells. We measured a significant, proportional increase in the total number of nucleated bone marrow precursor cells between the 1 and 5 mL samples (mean±SD, 27±13 and 152±78 million nucleated cells, respectively; P<0.0001). When the aspiration volume doubled from 5 to 10 mL the total number of nucleated cells was 178±76 million (P=0.17). A proportional increase from 2214 alkaline phosphatase-positive colony-forming units in the 1 mL sample to 14,100 alkaline phosphatase-positive colony-forming units in the 5 mL sample was observed. However, the number of colony-forming units per aspirate decreased to 11,880 in the 10 mL sample. These data demonstrate that in children aspiration up to 5 mL bone marrow from the iliac crest yields a proportional increase in osteoblastic progenitor cells per aspirate. Increasing the aspiration volume beyond 5 mL results in hemodilution, rather than further selection of osteoblastic material. These data provide clinicians with a guideline for optimizing aspiration volume of bone marrow in

  8. Generation of clinical grade human bone marrow stromal cells for use in bone regeneration.

    PubMed

    Robey, Pamela G; Kuznetsov, Sergei A; Ren, Jiaqiang; Klein, Harvey G; Sabatino, Marianna; Stroncek, David F

    2015-01-01

    In current orthopaedic practice, there is a need to increase the ability to reconstruct large segments of bone lost due to trauma, resection of tumors and skeletal deformities, or when normal regenerative processes have failed such as in non-unions and avascular necrosis. Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells), when used in conjunction with appropriate carriers, represent a means by which to achieve bone regeneration in such cases. While much has been done at the bench and in pre-clinical studies, moving towards clinical application requires the generation of clinical grade cells. What is described herein is an FDA-approved cell manufacturing procedure for the ex vivo expansion of high quality, biologically active human BMSCs. This article is part of a Special Issue entitled Stem Cells and Bone.

  9. Growth and adipogenic differentiation of mesenchymal stromal bone marrow cells during culturing in 3D macroporous agarose cryogel sponges.

    PubMed

    Petrenko, Yu A; Petrenko, A Yu; Damshkaln, L G; Volkova, N A; Lozinsky, V I

    2008-07-01

    We studied the possibility of population of macroporous agarose cryogel sponges by mesenchymal stromal bone marrow cells with their subsequent adipogenic differentiation. After 7-day culturing of mesenchymal stromal cells in agarose cryogel, the level of cell proliferation was 35%. After 3-week culturing in a medium inducing adipogenesis we observed accumulation of intracellular neutral lipids positively stained with Oil Red O. These findings can be used for the development of bioengineering constructions of the adipose tissue on the basis of spongy carriers.

  10. Bone morphogenetic protein-2 enhances the osteogenic differentiation capacity of mesenchymal stromal cells derived from human bone marrow and umbilical cord

    PubMed Central

    Marupanthorn, Kulisara; Tantrawatpan, Chairat; Kheolamai, Pakpoom; Tantikanlayaporn, Duangrat; Manochantr, Sirikul

    2017-01-01

    Mesenchymal stromal cells (MSCs) are multipotent cells that can give rise to different cell types of the mesodermal lineages. They are powerful sources for cell therapy in regenerative medicine as they can be isolated from various tissues, and can be expanded and induced to differentiate into multiple lineages. Recently, the umbilical cord has been suggested as an alternative source of MSCs. Although MSCs derived from the umbilical cord can be induced to differentiate into osteoblasts with a phenotypic similarity to that of bone marrow-derived MSCs, the differentiation ability is not consistent. In addition, MSCs from the umbilical cord require a longer period of time to differentiate into osteoblasts. Previous studies have demonstrated the benefits of bone morphogenetic protein-2 (BMP-2) in bone tissue regeneration. In addition, several studies have supported the use of BMP-2 in periodontal regeneration, sinus lift bone-grafting and non-unions in oral surgery. Although the use of BMP-2 for bone tissue regeneration has been extensively investigated, the BMP-2-induced osteogenic differentiation of MSCs derived from the umbilical cord has not yet been fully examined. Therefore, in this study, we aimed to examine the effects of BMP-2 on the osteogenic differentiation of MSCs derived from umbilical cord compared to that of MSCs derived from bone marrow. The degree of osteogenic differentiation following BMP-2 treatment was determined by assessing alkaline phosphatase (ALP) activity, and the expression profiles of osteogenic differentiation marker genes, osterix (Osx), Runt-related transcription factor 2 (Runx2) and osteocalcin (Ocn). The results revealed that BMP-2 enhanced the osteogenic differentiation capacity of MSCs derived from both bone marrow and umbilical cord as demonstrated by increased ALP activity and the upregulation of osteogenic differentiation marker genes. The enhancement of the osteogenic differentiation capacity of MSCs by BMP-2 suggests that these

  11. [Analysis of sensitivity of stromal stem cells (CFU-f) from rat bone marrow and fetal liver to 5-fluorouracil].

    PubMed

    Paiushina, O V; Damaratskaia, E I; Bueverova, E I; Nikonova, T M; Butorina, N N; Molchanova, E A; Starostin, V I

    2006-01-01

    The sensitivity of stromal stem cells (CFU-f) from rat bone marrow and fetal liver to the cytotoxic effect of 5-fluorouracil (5-FU) was compared in vivo and in vitro. Cells from both tissues demonstrated a similar resistance to 5-FU in vitro; however, stromal stem cells from fetal liver proved notably more sensitive to 5-FU compared to marrow CFU-f in vivo. Cells forming colonies of different size were identified in stem cell populations from both tissues. Cells giving rise to small colonies had a higher resistance to 5-FU both in vivo and in vitro.

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

    PubMed

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

    2002-08-01

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

  13. Effects of the bifunctional sulfoxide MMS350, a radiation mitigator, on hematopoiesis in long-term bone marrow cultures and on radioresistance of marrow stromal cell lines.

    PubMed

    Shinde, Ashwin; Epperly, Michael W; Cao, Shaonan; Franicola, Darcy; Shields, Donna; Wang, Hong; Wipf, Peter; Sprachman, Melissa M; Greenberger, Joel S

    2014-01-01

    The ionizing irradiation mitigator MMS350 prolongs survival of mice treated with total-body irradiation and prevents radiation-induced pulmonary fibrosis when added to drinking water at day 100 after thoracic irradiation. The effects of MMS350 on hematopoiesis in long-term bone marrow culture and on the radiobiology of derived bone marrow stromal cell lines were tested. Long-term bone marrow cultures were established from C57BL/6NTac mice and maintained in a high-humidity incubator, with 7% CO2 and the addition of 100 μM MMS350 at the weekly media change. Over 10 weeks in culture, MMS350 had no significant effect on maintenance of hematopoietic stem cell production, or on nonadherent cells or colony-forming units of hematopoietic progenitor cells. Stromal cell lines derived from non MMS350-treated long-term cultures or control stromal cells treated with MMS350 were radioresistant in the clonogenic survival curve assay. MMS350 is a non-toxic, highly water-soluble radiation mitigator that exhibits radioprotective effects on bone marrow stromal cells.

  14. Enhanced accumulation of adipocytes in bone marrow stromal cells in the presence of increased extracellular and intracellular [Ca{sup 2+}

    SciTech Connect

    Hashimoto, Ryota; Katoh, Youichi; Nakamura, Kyoko; Itoh, Seigo; Iesaki, Takafumi; Daida, Hiroyuki; Nakazato, Yuji; Okada, Takao

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer High [Ca{sup 2+}]{sub o} enhances adipocyte accumulation in the presence of adipogenic inducers. Black-Right-Pointing-Pointer High [Ca{sup 2+}]{sub o} enhances both proliferation and adipocyte differentiation in BMSCs. Black-Right-Pointing-Pointer High [Ca{sup 2+}]{sub o} induces an increase in [Ca{sup 2+}]{sub o} in BMSCs. Black-Right-Pointing-Pointer An intracellular Ca{sup 2+} chelator suppresses the enhancement in adipocyte accumulation. Black-Right-Pointing-Pointer Controlling [Ca{sup 2+}]{sub o} may govern the balance of adipocyte and osteoblast development. -- Abstract: The bone marrow stroma contains osteoblasts and adipocytes that have a common precursor: the pluripotent mesenchymal stem cell found in bone marrow stromal cells (BMSCs). Local bone marrow Ca{sup 2+} levels can reach high concentrations due to bone resorption, which is one of the notable features of the bone marrow stroma. Here, we describe the effects of high [Ca{sup 2+}]{sub o} on the accumulation of adipocytes in the bone marrow stroma. Using primary mouse BMSCs, we evaluated the level of adipocyte accumulation by measuring Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity. High [Ca{sup 2+}]{sub o} enhanced the accumulation of adipocytes following treatment with both insulin and dexamethasone together but not in the absence of this treatment. This enhanced accumulation was the result of both the accelerated proliferation of BMSCs and their differentiation into adipocytes. Using the fura-2 method, we also showed that high [Ca{sup 2+}]{sub o} induces an increase in [Ca{sup 2+}]{sub i}. An intracellular Ca{sup 2+} chelator suppressed the enhancement in adipocyte accumulation due to increased [Ca{sup 2+}]{sub o} in BMSCs. These data suggest a new role for extracellular Ca{sup 2+} in the bone marrow stroma: increased [Ca{sup 2+}]{sub o} induces an increase in [Ca{sup 2+}]{sub i} levels, which in turn enhances the accumulation of

  15. Engraftment and regenerative effects of bone marrow stromal cell transplantation on damaged rat olfactory mucosa.

    PubMed

    Kwon, Jang-Woo; Jo, Hyo Gyeong; Park, Sang Man; Ku, Cheol Hyo; Park, Dong-Joon

    2016-09-01

    To develop a new therapeutic method to treat olfactory deficits, we investigated the engraftment and regenerative effects of transplanted bone marrow stromal cells (BMSCs) on damaged rat olfactory mucosa. To induce olfactory nerve degeneration, one side of the olfactory mucosa of Sprague-Dawley rats was damaged via Triton X-100 irrigation. Phosphate-buffered saline containing syngeneic BMSCs was injected into the olfactory mucosa for transplantation. PKH fluorescent cell dye labeling of BMSCs was used to monitor the transplanted cells. After transplantation of BMSCs, the thickness and regeneration of olfactory mucosa were analyzed using hematoxylin-eosin (H&E) staining. S100 immunohistochemical staining was used to measure nerve sheath regeneration. The increase in NGF (nerve growth factor) level in the olfactory mucosa was measured by Western blot analysis. Transplanted bone marrow stromal cells were engrafted to the lamia propria of damaged mucosa. The mean time for normalization of thickness and morphological recovery of the olfactory mucosa was 4 weeks in the therapeutic group and 9 weeks in the control group. S100 immunoreactivity was higher on the BMSC-treated side than on the control side. During regeneration, the expression of NGF increased in the olfactory mucosa of the experimental group. Based on these results, BMSC transplantation accelerated regeneration of olfactory mucosa damaged by Triton X-100, and NGF may be essential to this regenerative process.

  16. The Effects of Fungal Volatile Organic Compounds on Bone Marrow Stromal Cells

    PubMed Central

    Hokeness, Kirsten; Lux, Hillary; Kratch, Jaqueline; Nadolny, Christina; Aicardi, Kristie; Reid, Christopher

    2015-01-01

    Evidence has shown that individuals exposed to indoor toxic molds for extended periods of time have elevated risk of developing numerous respiratory illnesses and certain types of cancer. It is not clear at the cellular level, what impact mold exposure has on the immune system. Herein we show that two fungal volatiles (E)-2-octenal and oct-1-en-3-ol have cytotoxic effects on murine bone marrow stromal (BMS) cells. To further analyze alterations to the cell, we evaluated the impact these VOCs have on membrane composition and hence fluidity. Both (E)-2-octenal and oct-1-en-3-ol exposure caused a shift to unsaturated fatty acids and lower cholesterol levels in the membrane. This indicates that the volatile organic compounds (VOCs) under investigation increased membrane fluidity. These vast changes to the cell membrane are known to contribute to the breakdown of normal cell function and possibly lead to death. Since bone marrow stromal cells are vital for the appropriate development and activation of immune cells, this study provides the foundation for understanding the mechanism at a cellular level for how mold exposure can lead to immune-related disease conditions. PMID:24392920

  17. [An experimental study on biocompatibility of Bio-oss collagen with cultured bone marrow stromal cells].

    PubMed

    Song, Zhong-Chen; Shu, Rong; Xie, Yu-Feng; Zhang, Xiu-Li; Yin, De-Min

    2008-04-01

    To study the biocompatibility of Bio-oss collagen with cultured bone marrow stromal cells. The bone marrow stromal cells (BMSCs) of rhesus monkey were cultured with Bio-oss collagen in vitro. Cell attachment was observed with laser confocal microscope. Cell proliferation rates were assessed with MTT assay. ALP activity was also detected. The data was statistically analyzed with SAS6.12 software package for Student's t test. The rhesus BMSCs could attach to the surface of Bio-oss collagen. In cell proliferation rates, there was no significant difference between the control group and experimental group at 2-day and 5-day(P>0.05). However, at 8-day, the difference was significant (P<0.05). For ALP activity, there was no significant difference among different time point (P>0.05). The Bio-oss collagen has good biocompatibility with rhesus BMSCs, and could be used to repair periodontal bone defects as a biomaterial in tissue engineering.

  18. Cell interactions between hematopoietic and stromal cells in the embryonic chick bone marrow.

    PubMed

    Sorrell, J M; Weiss, L

    1980-05-01

    Light microscopic, scanning electron microscopic, and transmission electron microscopic studies of the early developmental stages of chick embryonic bone marrow disclose characteristic associations of the first hematopoietic cells with stromal cells. The first hematopoietic cells, large basophilic cells that we have termed presumptive stem cells, segregate into erythropoietic and granulopoietic regions. Intravascular erythropoietic cells associate with sinusoidal endothelial cells, while granulopoietic cells associate with extravascular reticular cells. Extensive, intimate contacts between erythroid and endothelial cells are maintained, in part, by marginal arrays of microtubules, which promote a flattening of the adherent erythroid cell surface. In addition, cell surface components of opposing cells, visualized by ruthenium red staining, appear to merge and possibly to interact. Granulopoietic cells establish intimate but less extensive associations with reticular cells through cell-surface interactions. Stationary granuloid cells appear to be held in place by small, thin processes emanating from the sheet-like reticular cells. Granuloid cells are capable of moving within the extravascular region, using reticular cell surfaces as a substrate. Intimate associations also occur among granulopoietic cells, the significance of which is unclear. Thus, sinusoidal endothelial cells and reticular cells comprise the critical non-hematopoietic or stromal elements of avian bone marrow, where they have a putative role in segregating presumptive stem cells into erythrocyteic and granulocytic compartments. They serve as an architectual, and possibly regulatory, framework on which hematopoiesis occurs.

  19. Silicate bioceramics enhanced vascularization and osteogenesis through stimulating interactions between endothelia cells and bone marrow stromal cells.

    PubMed

    Li, Haiyan; Xue, Ke; Kong, Ni; Liu, Kai; Chang, Jiang

    2014-04-01

    The facts that biomaterials affect the behavior of single type of cells have been widely accepted. However, the effects of biomaterials on cell-cell interactions have rarely been reported. Bone tissue engineering involves osteoblastic cells (OCs), endothelial cells (ECs) and the interactions between OCs and ECs. It has been reported that silicate biomaterials can stimulate osteogenic differentiation of OCs and vascularization of ECs. However, the effects of silicate biomaterials on the interactions between ECs and OCs during vascularization and osteogenesis have not been reported, which are critical for bone tissue regeneration in vivo. Therefore, this study aimed to investigate the effects of calcium silicate (CS) bioceramics on interactions between human umbilical vein endothelial cells (HUVECs) and human bone marrow stromal cells (HBMSCs) and on stimulation of vascularization and osteogenesis in vivo through combining co-cultures with CS containing scaffolds. Specifically, the effects of CS on the angiogenic growth factor VEGF, osteogenic growth factor BMP-2 and the cross-talks between VEGF and BMP-2 in the co-culture system were elucidated. Results showed that CS stimulated co-cultured HBMSCs (co-HBMSCs) to express VEGF and the VEGF activated its receptor KDR on co-cultured HUVECs (co-HUVECs), which was also up-regulated by CS. Then, BMP-2 and nitric oxide expression from the co-HUVECs were stimulated by CS and the former stimulated osteogenic differentiation of co-HBMSCs while the latter stimulated vascularization of co-HVUECs. Finally, the poly(lactic-co-glycolic acid)/CS composite scaffolds with the co-cultured HBMSCs and HUVECs significantly enhanced vascularization and osteogenic differentiation in vitro and in vivo, which indicates that it is a promising way to enhance bone regeneration by combining scaffolds containing silicate bioceramics and co-cultures of ECs and OCs.

  20. Initial Binding and Recellularization of Decellularized Mouse Lung Scaffolds with Bone Marrow-Derived Mesenchymal Stromal Cells

    PubMed Central

    Daly, Amanda B.; Wallis, John M.; Borg, Zachary D.; Bonvillain, Ryan W.; Deng, Bin; Ballif, Bryan A.; Jaworski, Diane M.; Allen, Gilman B.

    2012-01-01

    Recellularization of whole decellularized lung scaffolds provides a novel approach for generating functional lung tissue ex vivo for subsequent clinical transplantation. To explore the potential utility of stem and progenitor cells in this model, we investigated recellularization of decellularized whole mouse lungs after intratracheal inoculation of bone marrow-derived mesenchymal stromal cells (MSCs). The decellularized lungs maintained structural features of native lungs, including intact vasculature, ability to undergo ventilation, and an extracellular matrix (ECM) scaffold consisting primarily of collagens I and IV, laminin, and fibronectin. However, even in the absence of intact cells or nuclei, a number of cell-associated (non-ECM) proteins were detected using mass spectroscopy, western blots, and immunohistochemistry. MSCs initially homed and engrafted to regions enriched in types I and IV collagen, laminin, and fibronectin, and subsequently proliferated and migrated toward regions enriched in types I and IV collagen and laminin but not provisional matrix (fibronectin). MSCs cultured for up to 1 month in either basal MSC medium or in a small airways growth media (SAGM) localized in both parenchymal and airway regions and demonstrated several different morphologies. However, while MSCs cultured in basal medium increased in number, MSCs cultured in SAGM decreased in number over 1 month. Under both media conditions, the MSCs predominantly expressed genes consistent with mesenchymal and osteoblast phenotype. Despite a transient expression of the lung precursor TTF-1, no other airway or alveolar genes or vascular genes were expressed. These studies highlight the power of whole decellularized lung scaffolds to study functional recellularization with MSCs and other cells. PMID:21756220

  1. Asperosaponin VI promotes bone marrow stromal cell osteogenic differentiation through the PI3K/AKT signaling pathway in an osteoporosis model

    PubMed Central

    Ke, Ke; Li, Qi; Yang, Xiaofeng; Xie, Zhijian; Wang, Yu; Shi, Jue; Chi, Linfeng; Xu, Weijian; Hu, Lingling; Shi, Huali

    2016-01-01

    Asperosaponin VI (ASA VI), a natural compound isolated from the well-known traditional Chinese herb Radix Dipsaci, has an important role in promoting osteoblast formation. However, its effects on osteoblasts in the context of osteoporosis is unknown. This study aimed to investigate the effects and mechanism of ASA VI action on the proliferation and osteogenic differentiation of bone marrow stromal cells isolated from the ovariectomized rats (OVX rBMSCs). The toxicity of ASA VI and its effects on the proliferation of OVX rBMSCs were measured using a CCK-8 assay. Various osteogenic differentiation markers were also analyzed, such as ALP activity, calcified nodule formation, and the expression of osteogenic genes, i.e., ALP, OCN, COL 1 and RUNX2. The results indicated that ASA VI promoted the proliferation of OVX rBMSCs and enhanced ALP activity and calcified nodule formation. In addition, while ASA VI enhanced the expression of ALP, OCN, Col 1 and RUNX2, treatment with LY294002 reduced all of these osteogenic effects and reduced the p-AKT levels induced by ASA VI. These results suggest that ASA VI promotes the osteogenic differentiation of OVX rBMSCs by acting on the phosphatidylinositol—3 kinase/AKT signaling pathway. PMID:27756897

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

    SciTech Connect

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

    2007-03-09

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

  3. Bone marrow regeneration promoted by biophysically sorted osteoprogenitors from mesenchymal stromal cells.

    PubMed

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

    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.

  4. MicroRNA-23a mediates post-transcriptional regulation of CXCL12 in bone marrow stromal cells

    PubMed Central

    Arabanian, Laleh S.; Fierro, Fernando A.; Stölzel, Friedrich; Heder, Carolin; Poitz, David M.; Strasser, Ruth H.; Wobus, Manja; Borhäuser, Martin; Ferrer, Ruben A.; Platzbecker, Uwe; Schieker, Matthias; Docheva, Denitsa; Ehninger, Gerhard; Illmer, Thomas

    2014-01-01

    The chemokine CXCL12 regulates the interaction between hematopoietic stem and progenitor cells and bone marrow stromal cells. Although its relevance in the bone marrow niche is well recognized, the regulation of CXCL12 by microRNA is not completely understood. We transfected a library of 486 microRNA in the bone marrow stromal cell line SCP-1 and studied the expression of CXCL12. Twenty-seven microRNA were shown to downregulate expression of CXCL12. Eight microRNA (miR-23a, 130b, 135, 200b, 200c, 216, 222, and 602) interacted directly with the 3′UTR of CXCL12. Next, we determined that only miR-23a is predicted to bind to the 3′UTR and is strongly expressed in primary bone marrow stromal cells. Modulation of miR-23a changes the migratory potential of hematopoietic progenitor cells in co-culture experiments. We discovered that TGFB1 mediates its inhibitory effect on CXCL12 levels by upregulation of miR-23a. This process was partly reversed by miR-23a molecules. Finally, we determined an inverse expression of CXCL12 and miR-23a in stromal cells from patients with myelodys-plastic syndrome indicating that the interaction has a pathophysiological role. Here, we show for the first time that CXCL12-targeting miR23a regulates the functional properties of the hematopoietic niche. PMID:24584347

  5. Use of autologous bone marrow mononuclear cells and cultured bone marrow stromal cells in dogs with orthopaedic lesions.

    PubMed

    Crovace, A; Favia, A; Lacitignola, L; Di Comite, M S; Staffieri, F; Francioso, E

    2008-09-01

    The aim of the study is to evaluate the clinical application in veterinary orthopedics of bone marrow mononuclear cells (BMMNCs) and cultured bone marrow stromal cells (cBMSCs) for the treatment of some orthopaedic lesions in the dog. The authors carried out a clinical study on 14 dogs of different breed, age and size with the following lesions: 1 bone cyst of the glenoid rime; 2 nonunion of the tibia; 3 nonunion of the femur; 2 lengthening of the radius; 1 large bone defect of the distal radius;1 nonunion with carpus valgus; 4 Legg-Calvé-Perthés disease. In 9 cases the BMMCNs were used in combination with a three dimensional resorbable osteogenic scaffold the chemical composition and size of which facilitates the ingrowth of bone. In these cases the BMMNCs were suspended in an adequate amount of fibrin glue and then distribuited uniformly on a Tricalcium-Phosphate (TCP) scaffold onto which were also added some drops of thrombin. In 1 case of nonunion of the tibia and in 3 cases of Legg-Calvè-Perthés (LCP) disease the cultured BMSCs were used instead because of the small size of the dogs and of the little amount of aspirated bone marrow. X-ray examinations were performed immediately after the surgery. Clinical, ultrasounds and X-ray examinations were performed after 20 days and then every month. Until now the treated dogs have shown very good clinical and X-ray results. One of the objectives of the study was to use the BMMNCs in clinical application in orthopaedic lesions in the dog. The advantages of using the cells immediately after the bone marrow is collected, are that the surgery can be performed the same day, the cells do not need to be expanded in vitro, they preserve their osteogenic potential to form bone and promote the proper integration of the implant with the bone and lastly, the technique is easier and the costs are lower.

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

    SciTech Connect

    Hashimoto, Ryota; Katoh, Youichi; Miyamoto, Yuki; Itoh, Seigo; Daida, Hiroyuki; Nakazato, Yuji; Okada, Takao

    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. Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells

    PubMed Central

    Kalinkovich, Alexander; Itkin, Tomer; Ludin, Aya; Kao, Wei-Ming; Battista, Michela; Tesio, Melania; Kollet, Orit; Cohen, Neta Netzer; Margalit, Raanan; Buss, Eike C.; Baleux, Francoise; Oishi, Shinya; Fujii, Nobutaka; Larochelle, Andre; Dunbar, Cynthia E.; Broxmeyer, Hal E.; Frenette, Paul S.; Lapidot, Tsvee

    2014-01-01

    Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine SDF-1 to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady-state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4+ human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, while β2-adrenergic antagonist inhibited both steady-state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes. PMID:21494253

  8. [Heterotopic osteogenesis of autogenous marrow stromal cells on ceramic bovine bone/ hydrogel scaffold].

    PubMed

    He, Dawei; Jin, Yan; Luo, Kai; Li, Shibao

    2006-02-01

    To observe the heterotopic osteogenesis of the autogenous marrow stromal cells (MSCs) on the ceramic bovine bone (CBB)/hydrogel scaffold (HG) and the effects of the recombinant human bone morphogenetic protein-2 (rhBMP-2) and the transforming growth factor beta (TGF-beta) on osteogenesis. The autogenous marrow stromal cells were cultured by the mineralized condition medium (10% FBS, dexamethasone 10 nmol, L-vitamin C 50 mg/L, beta-sodium glycerophosphate DMEM culture medium 10 mmol). At 5 days, the MSCs differentiation was observed by Type I collagen, the Mend calcium-cobalt staining, and the Von-Kossa staining. The cell suspension of 5 x 10(6)/ml was obtained. There were three groups: Group A: added in rhBMP-2 (10 microg)-TGF-beta (0.05 microg); Group B: added in TGF-beta (0.05 microg); and Group C (the control group): without the growth factor. Then, the MSCs loading on CBB/HG were embedded in the autogenous subcutaneous area at 4 and 8 weeks, and the osteogenesis was observed by the HE staining and the modified Mallory's trichrome staining, with an image analysis. Type I collagen and the bone morphogenetic synthesis were examined by the immunohistochemistry stains. Most MSCs induced by the mineralized condition medium at 5 days became smaller and polygon-shaped, and the cytodendrite became shorter. The MSCs were observed by the Mend calcium-cobalt staining. Some brown and black grains were found in the cytochylema. The MSCs were positive for the Type I collagen immunohistochemistry stains. At 20 days, the mineralized nubs were found by the Von Kossas stains. At 4 weeks, some strips of the new bone were observed by the HE staining and the modified Mallory's trichrome staining in all the groups. The bone matrix area was significantly larger in Group A than in Group B(P < 0.01). The average gray degrees of Type I collagen were lower in Groups A and B than in Group C. However, there was no significant difference in the bone morphogenesis among the three groups. At 8

  9. Transplantation of goat bone marrow stromal cells to the degenerating intervertebral disc in a goat disc-injury model

    PubMed Central

    Zhang, Yejia; Drapeau, Susan; An, Howard S.; Thonar, Eugene J-M.A.; Anderson, D. Greg

    2010-01-01

    Study Design In vivo randomized controlled study in the goat intervertebral disc (IVD) injury model. Objective To define the effects of allogeneic bone marrow-derived stromal cell injected into the degenerating goat IVDs. Summary of Background Data Transplantation of bone marrow stromal cells to the degenerating disc has been suggested as a means to correct the biologic incompetence of the disc. However, large animal models with IVDs similar in shape and size to those of humans are needed to define the efficacy and safety of this approach. Methods Goat IVD degeneration was induced by stabbing with a #15 blade. One month after disc injury, the injured discs were randomly selected to receive goat bone marrow-derived stromal cell (suspended in hydrogel), saline (control), or hydrogel (control) injections. Three and 6 months after stem cell transplantation, goats were euthanized and the IVD were examined for biochemical content and tissue morphology. MR images at 3- and 6-month time points were also examined. Results The goat large animal model shows early degenerative changes following disc injury. Degenerating IVDs injected with bone marrow stromal cells showed significantly increased proteoglycan (PG) accumulation within their nucleus pulposus (NP) region. However, collagen content, MRI grade and histology did not show statistically significant differences between the cell-treated and control IVDs. Conclusions Following transplantation of bone marrow stromal cells, NP tissue contained more PG than control discs. Although this result was promising, the rate and severity of degeneration in this goat disc injury were modest, suggesting that a more severe injury and a larger sample size is indicated for future studies to better define the utility of cell therapies in this model. PMID:20890267

  10. Age- and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic progenitors.

    PubMed

    Muschler, G F; Nitto, H; Boehm, C A; Easley, K A

    2001-01-01

    Bone marrow harvested by aspiration contains connective tissue progenitor cells which can be induced to express a bone phenotype in vitro. The number of osteoblastic progenitors can be estimated by counting the colony-forming units which express alkaline phosphatase (CFU-APs). This study was undertaken to test the hypothesis that human aging is associated with a significant change in the number or prevalence of osteoblastic progenitors in the bone marrow. Four 2-ml bone marrow aspirates were harvested bilaterally from the anterior iliac crest of 57 patients, 31 men (age 15-83) and 26 women (age 13-79). A mean of 64 million nucleated cells was harvested per aspirate. The mean prevalence of CFU-APs was found to be 55 per million nucleated cells. These data revealed a significant age-related decline in the number of nucleated cells harvested per aspirate for both men and women (P = 0.002). The number of CFU-APs harvested per aspirate also decreased significantly with age for women (P = 0.02), but not for men (P = 0.3). These findings are relevant to the harvest of bone marrow derived connective tissue progenitors for bone grafting and other tissue engineering applications, and may also be relevant to the pathophysiology of age-related bone loss and post-menopausal osteoporosis.

  11. Bone marrow mesenchymal stromal cells induce nitric oxide synthase-dependent differentiation of CD11b+ cells that expedite hematopoietic recovery.

    PubMed

    Trento, Cristina; Marigo, Ilaria; Pievani, Alice; Galleu, Antonio; Dolcetti, Luigi; Wang, Chun-Yin; Serafini, Marta; Bronte, Vincenzo; Dazzi, Francesco

    2017-02-09

    Bone marrow microenvironment is fundamental for hematopoietic homeostasis. Numerous efforts have been made to reproduce or manipulate its activity to facilitate engraftment after hematopoietic stem cell transplantation but clinical results remain unconvincing. This probably reflects the complexity of the hematopoietic niche. Recent data have demonstrated the fundamental role of stromal and myeloid cells in regulating hematopoietic stem cell self-renewal and mobilization in the bone marrow. In this study we unveil a novel interaction by which bone marrow mesenchymal stromal cells induce the rapid differentiation of CD11b+ myeloid cells from bone marrow progenitors. Such an activity requires the expression of nitric oxide synthase-2. Importantly, the administration of these mesenchymal stromal cells-educated CD11b+ cells accelerates hematopoietic reconstitution in bone marrow transplant recipients. We conclude that the liaison between mesenchymal stromal cells and myeloid cells is fundamental in hematopoietic homeostasis and suggests that it can be harnessed in clinical transplantation.

  12. [Reorganization of actin cytoskeleton in the initial stage of transendothelial migration of bone marrow multipotent mesenchymal stromal cells].

    PubMed

    Aleksandrova, S A; Pinaev, G P

    2014-01-01

    The analysis of actin cytoskeleton reorganization in rat bone marrow multipotent mesenchymal stromal cells after one hour adhesion to a monolayer of endothelial cell line EA.hy 926 allowed us to identify three types of cells interacting with the endothelial cells. Approximately half of multipotent mesenchymal stromal cells retained a rounded shape, most of them contained large round actin aggregates, had irregular borders and contacted with the surface of the endothelial cells by microvilli or protrusions similar to small lamellae. Almost all other cells were surrounded by narrow lamellae along the entire perimeter. In addition, a small amount.of elongated flattened cells that contacting with endothelial cells by means of focal contacts was observed. Microenvironmental factors such as proinflammatory cytokine tumor necrosis factor α or plasma proteins affected the ratio of stromal cell types, with different types of organization of the actin cytoskeleton in multipotent mesenchymal stromal cells population.

  13. Intra-Bone Marrow Transplantation of Endosteal Bone Marrow Cells Facilitates Allogeneic Hematopoietic and Stromal Cells Engraftment Dependent on Early Expression of CXCL-12.

    PubMed

    Chen, Chen; Su, Yingjun; Chen, Jianwu; Zhang, Dongliang; Song, Yajuan; Guo, Shuzhong

    2015-09-16

    Hematopoietic stem cell transplantation (HSCT) has been considered as an effective approach at inducing allogeneic hematopoietic reconstitution and immune tolerance. However, it remains critical to find the optimal HSCT delivery method and robust sources of hematopoietic stem cells (HSCs). We introduced a new method by infusing allogeneic endosteal bone marrow cells (BMCs) harvested from long bones endosteum through intra-bone marrow transplantation (IBBMT) into irradiated mice. Recipient mice that were transplanted with central BMCs or through intravenous bone marrow transplantation (IVBMT) were used as controls (n=6 per group). We compared the new method with each control group for allogeneic HSCs homing pattern, peripheral blood chimerism level, skin allograft survival time, and donor stromal cell percentage in recipient BM. AMD3100 was injected to determine whether chemokine stromal cell-derived factor-1 (CXCL-12) was critical for the new method. More allogeneic HSCs homed into spleen and bone marrow for the new method as compared to each control group. IBBMT of endosteal BMCs led to a higher peripheral blood chimerism and skin allograft survival. At 18 weeks, donor stromal cell percentage in recipient BMCs was higher for the new method than in each control group. By AMD3100 blockade at day 1, peripheral blood chimerism level and donor stromal cell percentage were significantly reduced as compared to the control group without AMD3100 blockade. Our study suggests that IBBMT of endosteal BMCs is an effective approach for HSCT in inducing allogeneic hematopoietic reconstitution. The advantage is dependent upon the early expression of CXCL-12 after bone marrow transplantation.

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

    PubMed Central

    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. Organotins are potent activators of PPARγ and adipocyte differentiation in bone marrow multipotent mesenchymal stromal cells.

    PubMed

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

    2011-08-01

    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 10-20 nM) 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.

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

    PubMed

    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.

  17. Differences in xenobiotic detoxifying activities between bone marrow stromal cells from mice and rats: Implications for benzene-induced hematotoxicity

    SciTech Connect

    Zhu, Hong; Li, Yunbo; Trush, M.A.

    1995-10-01

    benzene is a human carcinogen; exposure can result in aplastic anemia and leukemia. Data from animal models are frequently used in benzene risk assessment. In rodent studies, mice are more sensitive to benzene-induced hematotoxicity than rats. Bone marrow stromal cells from mice were significantly more susceptible to the cytotoxicity induced by the benzene metabolites hydroquinone (HQ) and benzoquinone (BQ) than cells from rats. Since cellular gluthathione (GSH) and quinone reductase (QR) are known to play critical roles in modulating HQ-induced cytotoxicity, the GSH content and the QR and glutathione S-transferase (GST) activity in stromal cells from both species was measured. In rat cells, the GSH content and the QR specific activity were 2 and 28 times as much as those from mice, respectively. GSH and QR in both mouse and rat stromal cells were inducible by 1,2-dithiole-3-thione (D3T). D3T pretreatment of both mouse and rat stromal cells resulted in a marked protection against HQ-induced toxicity. Pretreatment of both mouse and rat stromal cells with GSH ethyl ester also provided a dramatic protection against HQ-induced toxicity. Conversely, dicoumarol, an inhibitor of QR, enhanced the HQ-induced toxicity in stromal cells from both mice and rats, indicating an important role for QR in modulating HQ-induced stromal toxicity. Buthionine sulfoximine (BSO), which depleted GSH significantly in both species, potentiated the HQ-induced toxicity in mouse but not in rat stromal cells. Surprisingly, incubation of stromal cells with BSO resulted in a significant induction of QR, especially in rats. Overall, this study demonstrates that the differences in stromal cellular GSH content and QR activity between mice and rats contribute to their respective susceptibility to HQ-induced cytotoxicity in vitro, and may be involved in the greater in vivo sensitivity of mice to benzene-induced hematotoxicity. 51 refs., 9 figs., 1 tab.

  18. Comparison of manual and automated cultures of bone marrow stromal cells for bone tissue engineering.

    PubMed

    Akiyama, Hirokazu; Kobayashi, Asako; Ichimura, Masaki; Tone, Hiroshi; Nakatani, Masaru; Inoue, Minoru; Tojo, Arinobu; Kagami, Hideaki

    2015-11-01

    The development of an automated cell culture system would allow stable and economical cell processing for wider clinical applications in the field of regenerative medicine. However, it is crucial to determine whether the cells obtained by automated culture are comparable to those generated by manual culture. In the present study, we focused on the primary culture process of bone marrow stromal cells (BMSCs) for bone tissue engineering and investigated the feasibility of its automation using a commercially available automated cell culture system in a clinical setting. A comparison of the harvested BMSCs from manual and automated cultures using clinically acceptable protocols showed no differences in cell yields, viabilities, surface marker expression profiles, and in vivo osteogenic abilities. Cells cultured with this system also did not show malignant transformation and the automated process was revealed to be safe in terms of microbial contamination. Taken together, the automated procedure described in this report provides an approach to clinical bone tissue engineering.

  19. Expression of brain natriuretic peptide by human bone marrow stromal cells.

    PubMed

    Song, S; Kamath, S; Mosquera, D; Zigova, T; Sanberg, P; Vesely, D L; Sanchez-Ramos, J

    2004-01-01

    Bone marrow stromal cells (BMSC) have been shown to generate neural cells under experimental conditions in vitro and following transplantation into animal models of stroke and traumatic CNS injury. Hastened recovery from the neurological deficit has not correlated with structural repair of the lesion in the stroke model. Secretory functions of BMSC, such as the elaboration of growth factors and cytokines, have been hypothesized to play a role in the enhanced recovery of neurological function. Using gene expression arrays, real time RT-PCR and radioimmunoassay, we have found that brain natriuretic peptide (BNP) is synthesized and released by BMSC at physiologically relevant levels in vitro. BNP, like its close homolog atrial natriuretic peptide (ANP), exerts powerful natriuretic, diuretic and vasodilatory effects. We speculate that transplanted BMSCs facilitate recovery from brain and spinal cord lesions by releasing BNP and other vasoactive factors that reduce edema, decrease intracranial pressure and improve cerebral perfusion.

  20. Polydatin induces bone marrow stromal cells migration by activation of ERK1/2.

    PubMed

    Chen, ZhenQiu; Wei, QiuShi; Hong, GuoJu; Chen, Da; Liang, Jiang; He, Wei; Chen, Mei Hui

    2016-08-01

    Bone marrow stromal cells (BMSCs) have proven to be useful for the treatment of numerous human diseases. However, the reparative ability of BMSCs is limited by their poor migration. Polydatin, widely used in traditional Chinese remedies, has proven to exert protective effects to BMSCs. However, little is known about its role in BMSCs migration. In this study, we studied the effects of polydatin on rat BMSCs migration using the scratch wound healing and transwell migration assays. Our results showed polydatin could promote BMSCs migration. Further experiments showed activation of ERK 1/2, but not JNK, was required for polydatin-induced BMSCs migration, suggesting that polydatin may promote BMSCs migration via the ERK 1/2 signaling pathways. Taken together, our results indicate that polydatin might be beneficial for stem cell replacement therapy by improving BMSCs migration.

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

    PubMed

    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.

  2. Correlation between nicotine-induced inhibition of hematopoiesis and decreased CD44 expression on bone marrow stromal cells.

    PubMed

    Khaldoyanidi, S; Sikora, L; Orlovskaya, I; Matrosova, V; Kozlov, V; Sriramarao, P

    2001-07-15

    This study demonstrates that in vivo exposure to cigarette smoke (CS) and in vitro treatment of long-term bone marrow cultures (LTBMCs) with nicotine, a major constituent of CS, result in inhibition of hematopoiesis. Nicotine treatment significantly delayed the onset of hematopoietic foci and reduced their size. Furthermore, the number of long-term culture-initiating cells (LTC-ICs) within an adherent layer of LTBMCs was significantly reduced in cultures treated with nicotine. Although the production of nonadherent mature cells and their progenitors in nicotine-treated LTBMCs was inhibited, this treatment failed to influence the proliferation of committed hematopoietic progenitors when added into methylcellulose cultures. Bone marrow stromal cells are an integral component of the hematopoietic microenvironment and play a critical role in the regulation of hematopoietic stem cell proliferation and self-renewal. Exposure to nicotine decreased CD44 surface expression on primary bone marrow-derived fibroblastlike stromal cells and MS-5 stromal cell line, but not on hematopoietic cells. In addition, mainstream CS altered the trafficking of hematopoietic stem/progenitor cells (HSPC) in vivo. Exposure of mice to CS resulted in the inhibition of HSPC homing into bone marrow. Nicotine and cotinine treatment resulted in reduction of CD44 surface expression on lung microvascular endothelial cell line (LEISVO) and bone marrow-derived (STR-12) endothelial cell line. Nicotine treatment increased E-selectin expression on LEISVO cells, but not on STR-12 cells. These findings demonstrate that nicotine can modulate hematopoiesis by affecting the functions of the hematopoiesis-supportive stromal microenvironment, resulting in the inhibition of bone marrow seeding by LTC-ICs and interfering with stem cell homing by targeting microvascular endothelial cells.

  3. The tropism of neurally differentiated bone marrow stromal cells towards C6 glioma.

    PubMed

    Long, Qianfa; Liu, Weiping; Zhong, Jun; Yi, Xicai; Liu, Yang; Liu, Yuanyang; Yang, Yang; Han, Rui; Fei, Zhou

    2011-10-24

    Recent studies have indicated that bone marrow stromal cells (BMSCs) have significant tropism towards glioma which makes them play an important role in carrying genes/drugs to inhibit the growth of glioma as cell vehicles. But BMSCs may differentiate into neural cells under entocranial environment and few researches support the idea that neurally differentiated bone marrow stromal cells (N-D-BMSCs) still hold the capacity of migrating to the tumor sites. The aim of our study was to investigate the tropism of N-D-BMSCs towards C6 glioma. In vitro migration assay was employed by transwell co-culture system and Student's t-test analysis indicated that N-D-BMSCs had the significant tropism towards C6 glioma-conditioned medium (GCM) (P<0.01). Furthermore, the vascular endothelial growth factor (VEGF) bioactivity of the C6 GCM was neutralized by the anti-rat VEGF antibody and our data suggested that the VEGF from C6 GCM hold chemoattraction for N-D-BMSCs and some other cytokines from the C6 GCM may be responsible for the chemoattraction for N-D-BMSCs. In vivo migration assay was carried out with cells transplantation and one way ANOVA analysis indicated that the tropism of N-D-BMSCs towards C6 glioma sites presented time variation (P-value=2.9E-20). Moreover, multiple comparisons for the time variables with the Student's t-test and the results suggested that the migration capacity of N-D-BMSCs towards C6 glioma sites reach the peak on the 7th day after transplantation. These results demonstrate that N-D-BMSCs as well as BMSCs have significant tropism towards C6 glioma. Published by Elsevier Ireland Ltd.

  4. Intrathecal injection of bone marrow stromal cells attenuates neurologic injury after spinal cord ischemia.

    PubMed

    Shi, Enyi; Kazui, Teruhisa; Jiang, Xiaojing; Washiyama, Naoki; Yamashita, Katsushi; Terada, Hitoshi; Bashar, Abul Hasan Muhammad

    2006-06-01

    It has been shown that transplantation of bone marrow stromal cells (MSCs) into the ischemic brain improves functional outcome. We sought to investigate whether intrathecal injection of MSCs can attenuate neurologic injury of spinal cord ischemia. Rabbit MSCs were expanded in vitro and were pre-labeled with bromodeoxyuridine. Spinal cord ischemia was induced in rabbits by infrarenal aortic occlusion. Group A and control A were subjected to a 20-minute ischemia and the ischemic duration was extended to 30 minutes in group B and control B. Two days before spinal cord ischemia, 1 x 10(8) MSCs were intrathecally injected into groups A and B, whereas vehicle alone was injected into the control groups. Hind-limb motor function was assessed during a 14-day recovery period with Tarlov criteria, and then histologic examination was performed. Marrow stromal cells survived and engrafted into the spinal cord 2 days after transplantation, and more MSCs were found in the lumbar spinal cord (ischemic segment) than in the thoracic spinal cord (nonischemic segment) at 14 days. Compared with their respective control groups, Tarlov scores were significantly higher in both groups A and B (p < 0.05, group A vs control A, at 2, 7, and 14 days; p < 0.05, group B vs control B, at 1, 2, 7, and 14 days, respectively). The number of intact motor neurons was much higher in the two experimental groups (p < 0.01 vs the corresponding control groups, respectively). Intrathecal injection of MSCs attenuates ischemic injury of spinal cord.

  5. Androgen, Estrogen and the Bone Marrow Microenvironment

    DTIC Science & Technology

    2009-12-01

    vivo in osteoblasts, BM stromal cells, and endothelial cells. Primary human bone marrow stromal cell cultures secrete IGFBP5. In vitro, treatment of...immortalized prostate epithelial cells. Treatment of mice with DES for 3 weeks had a dramatic effect on the bone. 518 genes were upregulated by...DES by at least 1.5 fold with a false discovery rate of < 5%. The genes that are 5-fold or greater overexpressed after DES treatment are: procollagen

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

    PubMed Central

    Soares, P.B.; Jeremias, T.S.; Alvarez-Silva, M.; Licínio, M.A.; Santos-Silva, M.C.; Vituri, C.L.

    2012-01-01

    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. PMID:23011404

  7. Different Procoagulant Activity of Therapeutic Mesenchymal Stromal Cells Derived from Bone Marrow and Placental Decidua.

    PubMed

    Moll, Guido; Ignatowicz, Lech; Catar, Rusan; Luecht, Christian; Sadeghi, Behnam; Hamad, Osama; Jungebluth, Philipp; Dragun, Duska; Schmidtchen, Artur; Ringdén, Olle

    2015-10-01

    While therapeutic mesenchymal stromal/stem cells (MSCs) have usually been obtained from bone marrow, perinatal tissues have emerged as promising new sources of cells for stromal cell therapy. In this study, we present a first safety follow-up on our clinical experience with placenta-derived decidual stromal cells (DSCs), used as supportive immunomodulatory and regenerative therapy for patients with severe complications after allogeneic hematopoietic stem cell transplantation (HSCT). We found that DSCs are smaller, almost half the volume of MSCs, which may favor microvascular passage. DSCs also show different hemocompatibility, with increased triggering of the clotting cascade after exposure to human blood and plasma in vitro. After infusion of DSCs in HSCT patients, we observed a weak activation of the fibrinolytic system, but the other blood activation markers remained stable, excluding major adverse events. Expression profiling identified differential levels of key factors implicated in regulation of hemostasis, such as a lack of prostacyclin synthase and increased tissue factor expression in DSCs, suggesting that these cells have intrinsic blood-activating properties. The stronger triggering of the clotting cascade by DSCs could be antagonized by optimizing the cell graft reconstitution before infusion, for example, by use of low-dose heparin anticoagulant in the cell infusion buffer. We conclude that DSCs are smaller and have stronger hemostatic properties than MSCs, thus triggering stronger activation of the clotting system, which can be antagonized by optimizing the cell graft preparation before infusion. Our results highlight the importance of hemocompatibility safety testing for every novel cell therapy product before clinical use, when applied using systemic delivery.

  8. MEK5 suppresses osteoblastic differentiation

    SciTech Connect

    Kaneshiro, Shoichi; Otsuki, Dai; Yoshida, Kiyoshi; Yoshikawa, Hideki; Higuchi, Chikahisa

    2015-07-31

    Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein kinase (MAPK) family and is activated by its upstream kinase, MAPK kinase 5 (MEK5), which is a member of the MEK family. Although the role of MEK5 has been investigated in several fields, little is known about its role in osteoblastic differentiation. In this study, we have demonstrated the role of MEK5 in osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells and bone marrow stromal ST2 cells. We found that treatment with BIX02189, an inhibitor of MEK5, increased alkaline phosphatase (ALP) activity and the gene expression of ALP, osteocalcin (OCN) and osterix, as well as it enhanced the calcification of the extracellular matrix. Moreover, osteoblastic cell proliferation decreased at a concentration of greater than 0.5 μM. In addition, knockdown of MEK5 using siRNA induced an increase in ALP activity and in the gene expression of ALP, OCN, and osterix. In contrast, overexpression of wild-type MEK5 decreased ALP activity and attenuated osteoblastic differentiation markers including ALP, OCN and osterix, but promoted cell proliferation. In summary, our results indicated that MEK5 suppressed the osteoblastic differentiation, but promoted osteoblastic cell proliferation. These results implied that MEK5 may play a pivotal role in cell signaling to modulate the differentiation and proliferation of osteoblasts. Thus, inhibition of MEK5 signaling in osteoblasts may be of potential use in the treatment of osteoporosis. - Highlights: • MEK5 inhibitor BIX02189 suppresses proliferation of osteoblasts. • MEK5 knockdown and MEK5 inhibitor promote differentiation of osteoblasts. • MEK5 overexpression inhibits differentiation of osteoblasts.

  9. Dissecting the role of bone marrow stromal cells on bone metastases.

    PubMed

    Buenrostro, Denise; Park, Serk In; Sterling, Julie A

    2014-01-01

    Tumor-induced bone disease is a dynamic process that involves interactions with many cell types. Once metastatic cancer cells reach the bone, they are in contact with many different cell types that are present in the cell-rich bone marrow. These cells include the immune cells, myeloid cells, fibroblasts, osteoblasts, osteoclasts, and mesenchymal stem cells. Each of these cell populations can influence the behavior or gene expression of both the tumor cells and the bone microenvironment. Additionally, the tumor itself can alter the behavior of these bone marrow cells which further alters both the microenvironment and the tumor cells. While many groups focus on studying these interactions, much remains unknown. A better understanding of the interactions between the tumor cells and the bone microenvironment will improve our knowledge on how tumors establish in bone and may lead to improvements in diagnosing and treating bone metastases. This review details our current knowledge on the interactions between tumor cells that reside in bone and their microenvironment.

  10. Amphiregulin-EGFR signaling mediates the migration of bone marrow mesenchymal progenitors toward PTH-stimulated osteoblasts and osteocytes.

    PubMed

    Zhu, Ji; Siclari, Valerie A; Liu, Fei; Spatz, Jordan M; Chandra, Abhishek; Divieti Pajevic, Paola; Qin, Ling

    2012-01-01

    Intermittent administration of parathyroid hormone (PTH) dramatically increases bone mass and currently is one of the most effective treatments for osteoporosis. However, the detailed mechanisms are still largely unknown. Here we demonstrate that conditioned media from PTH-treated osteoblastic and osteocytic cells contain soluble chemotactic factors for bone marrow mesenchymal progenitors, which express a low amount of PTH receptor (PTH1R) and do not respond to PTH stimulation by increasing cAMP production or migrating toward PTH alone. Conditioned media from PTH-treated osteoblasts elevated phosphorylated Akt and p38MAPK amounts in mesenchymal progenitors and inhibition of these pathways blocked the migration of these progenitors toward conditioned media. Our previous and current studies revealed that PTH stimulates the expression of amphiregulin, an epidermal growth factor (EGF)-like ligand that signals through the EGF receptor (EGFR), in both osteoblasts and osteocytes. Interestingly, conditioned media from PTH-treated osteoblasts increased EGFR phosphorylation in mesenchymal progenitors. Using several different approaches, including inhibitor, neutralizing antibody, and siRNA, we demonstrate that PTH increases the release of amphiregulin from osteoblastic cells, which acts on the EGFRs expressed on mesenchymal progenitors to stimulate the Akt and p38MAPK pathways and subsequently promote their migration in vitro. Furthermore, inactivation of EGFR signaling specifically in osteoprogenitors/osteoblasts attenuated the anabolic actions of PTH on bone formation. Taken together, these results suggest a novel mechanism for the therapeutic effect of PTH on osteoporosis and an important role of EGFR signaling in mediating PTH's anabolic actions on bone.

  11. Amphiregulin-EGFR Signaling Mediates the Migration of Bone Marrow Mesenchymal Progenitors toward PTH-Stimulated Osteoblasts and Osteocytes

    PubMed Central

    Zhu, Ji; Siclari, Valerie A.; Liu, Fei; Spatz, Jordan M.; Chandra, Abhishek; Divieti Pajevic, Paola; Qin, Ling

    2012-01-01

    Intermittent administration of parathyroid hormone (PTH) dramatically increases bone mass and currently is one of the most effective treatments for osteoporosis. However, the detailed mechanisms are still largely unknown. Here we demonstrate that conditioned media from PTH-treated osteoblastic and osteocytic cells contain soluble chemotactic factors for bone marrow mesenchymal progenitors, which express a low amount of PTH receptor (PTH1R) and do not respond to PTH stimulation by increasing cAMP production or migrating toward PTH alone. Conditioned media from PTH-treated osteoblasts elevated phosphorylated Akt and p38MAPK amounts in mesenchymal progenitors and inhibition of these pathways blocked the migration of these progenitors toward conditioned media. Our previous and current studies revealed that PTH stimulates the expression of amphiregulin, an epidermal growth factor (EGF)-like ligand that signals through the EGF receptor (EGFR), in both osteoblasts and osteocytes. Interestingly, conditioned media from PTH-treated osteoblasts increased EGFR phosphorylation in mesenchymal progenitors. Using several different approaches, including inhibitor, neutralizing antibody, and siRNA, we demonstrate that PTH increases the release of amphiregulin from osteoblastic cells, which acts on the EGFRs expressed on mesenchymal progenitors to stimulate the Akt and p38MAPK pathways and subsequently promote their migration in vitro. Furthermore, inactivation of EGFR signaling specifically in osteoprogenitors/osteoblasts attenuated the anabolic actions of PTH on bone formation. Taken together, these results suggest a novel mechanism for the therapeutic effect of PTH on osteoporosis and an important role of EGFR signaling in mediating PTH's anabolic actions on bone. PMID:23300521

  12. Treatment with bone marrow-derived stromal cells accelerates wound healing in diabetic rats.

    PubMed

    Kwon, David S; Gao, Xiaohua; Liu, Yong Bo; Dulchavsky, Deborah S; Danyluk, Andrew L; Bansal, Mona; Chopp, Michael; McIntosh, Kevin; Arbab, Ali S; Dulchavsky, Scott A; Gautam, Subhash C

    2008-06-01

    Bone marrow stem cells participate in tissue repair processes and may have a role in wound healing. Diabetes is characterised by delayed and poor wound healing. We investigated the potential of bone marrow-derived mesenchymal stromal cells (BMSCs) to promote healing of fascial wounds in diabetic rats. After manifestation of streptozotocin (STZ)-induced diabetic state for 5 weeks in male adult Sprague-Dawley rats, healing of fascial wounds was severely compromised. Compromised wound healing in diabetic rats was characterised by excessive polymorphonuclear cell infiltration, lack of granulation tissue formation, deficit of collagen and growth factor [transforming growth factor (TGF-beta), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor PDGF-BB and keratinocyte growth factor (KGF)] expression in the wound tissue and significant decrease in biomechanical strength of wounds. Treatment with BMSC systemically or locally at the wound site improved the wound-breaking strength (WBS) of fascial wounds. The improvement in WBS was associated with an immediate and significant increase in collagen levels (types I-V) in the wound bed. In addition, treatment with BMSCs increased the expression of growth factors critical to proper repair and regeneration of the damaged tissue moderately (TGF-beta, KGF) to markedly (EGF, VEGF, PDGF-BB). These data suggest that cell therapy with BMSCs has the potential to augment healing of the diabetic wounds.

  13. Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow

    SciTech Connect

    Itoh, K.; Tezuka, H.; Sakoda, H.; Konno, M.; Nagata, K.; Uchiyama, T.; Uchino, H.; Mori, K.J.

    1989-02-01

    Stromal cell lines, designated MS-1, -2, -3, -4, -5, -6, and -7 were established by irradiating the adherent cells in long-term bone marrow cultures with 900-rad x-rays. Two of the cell lines, MS-1 and MS-5, have the capacity to support the growth of hemopoietic stem cells (spleen colony-forming cells and granulocyte-macrophage colony-forming cells) for greater than 2 months in vitro. These two cell lines were alkaline phosphatase-, peroxidase-, and factor VIII-negative and positive for periodic acid-Schiff and nonspecific esterase. Extracellular matrix proteins such as fibronectin, laminin, and collagen type I were produced by these two cell lines. Neither MS-1 cell- nor MS-5 cell-conditioned medium supported the growth of hemopoietic stem cells, and hemopoietic stem cells were found preferentially to be under and on MS-1 and MS-5 layers rather than in suspension. Close contact with the MS-1 cell layer or the MS-5 cell layer appears to be essential in maintaining hemopoiesis in vitro. Conditioned media from MS-1 cells and MS-5 cells stimulated granulocyte colony formation from murine bone marrow cells in semisolid culture.

  14. Nicotinamide phosphoribosyltransferase (Nampt) may serve as the marker for osteoblast differentiation of bone marrow-derived mesenchymal stem cells.

    PubMed

    He, Xu; He, Jiaxue; Shi, Yingai; Pi, Chenchen; Yang, Yue; Sun, Yanan; Ma, Cao; Lin, Lin; Zhang, Lihong; Li, Yulin; Li, Yan

    2017-03-01

    Decreased bone volume and strength with aging and enhanced risk of fractures are in part due to reduced number of bone-forming mesenchymal stem cells (MSCs) and cellular dysfunction. In a previous study, we found that osteogenic differentiation of the multipotent and omnipotent preosteoblasts are accompanied by the alterations of intracellular NAD metabolism in which nicotinamide phosphoribosyltransferase (Nampt) plays a regulatory role. The increased Nampt during osteoblast differentiation, the enzyme catalyzing NAD resynthesis from nicotinamide was noted. However, whether Nampt will also be able to affect osteogenic differentiation of primary bone marrow-derived mesenchymal stem cells (BM-MSCs), it is still uncertain. Here we report the role of Nampt in regulating osteoblast differentiation in primary mouse BM-MSCs. We found that Nampt expression was progressively elevated during BM-MSCs osteogenic differentiation. The Nampt inhibitor FK866 or knock-down of Nampt in BM-MSCs led to declined osteoblastogenesis, including attenuated ALP activity, diminished matrix mineralization and down-regulated osteoblast specific marker genes. In addition, declined osteoblastogenesis by Nampt deficiency or addition of FK866 was related to lower intracellular NAD concentration and decreased Sirt1 activity. The present findings demonstrate that osteogenic differentiation in MSCs can be modulated by intracellular NAD metabolism, in which Nampt may serve as an applicable marker for the osteoblast determination.

  15. Paracrine Factors Produced by Bone Marrow Stromal Cells Induce Apoptosis and Neuroendocrine Differentiation in Prostate Cancer Cells

    PubMed Central

    Zhang, Chu; Soori, Mehrnoosh; Miles, Fayth; Sikes, Robert A.; Carson, Daniel D.; Chung, Leland L.W.; Farach-Carson, Mary C.

    2010-01-01

    Background Preferential bony metastasis of human prostate cancer (PCa) cells contributes to disease mortality and morbidity. Local factors in bone stromal extracellular matrix microenvironment affect tumor growth through paracrine interactions between tumor and stromal cells. Methods Using co-culture and medium transfer, we used several methods to assess interactions between PCa and bone stromal cells using three PCa cell lines: PC3, LNCaP, and the LNCaP derivative, C4-2B. Results Co-culture of LNCaP and C4-2B cells with bone marrow stromal cell lines, HS27a and HS5, decreased cell number, as did culture with conditioned medium (CM) harvested from these two cell lines suggesting a soluble paracrine factor was responsible. PC3 cell growth was unaffected. CM harvested from bone stromal cell lines triggered apoptosis in LNCaP and C4-2B cell lines, but not in PC3 cells. Surviving C4-2B cells grown in bone stromal cell CM over several days were growth arrested, suggesting presence of a growth inhibitor. Apoptosis induced by CM was dose-dependent. Flow cytometry demonstrated that over a five day culture period in stromal cell CM, LNCaP and C4-2B cell lines, but not PC3 cells, underwent greater apoptosis than parallel cultures in SF medium. The LNCaP and C4-2B cells showed morphology and biomarker expression consistent with transdifferentiation towards a neuroendocrine phenotype after exposure to stromal cell CM. Conclusions The reactive bone stromal microenvironment initially is hostile to PCa cells producing widespread apoptosis. Activation of transdifferentiation in a subset of apoptotic resistant cells may support phenotypic adaptation during disease progression in bone, eventually favoring lethal disease. PMID:20665531

  16. Evaluation of adipose-derived stromal vascular fraction or bone marrow-derived mesenchymal stem cells for treatment of osteoarthritis.

    PubMed

    Frisbie, David D; Kisiday, John D; Kawcak, Chris E; Werpy, Natasha M; McIlwraith, C Wayne

    2009-12-01

    The purpose of this study was the assessment of clinical, biochemical, and histologic effects of intraarticular administered adipose-derived stromal vascular fraction or bone marrow-derived mesenchymal stem cells for treatment of osteoarthritis. Osteoarthritis was induced arthroscopically in the middle carpal joint of all horses, the contralateral joint being sham-operated. All horses received treatment on Day 14. Eight horses received placebo treatment and eight horses received adipose-derived stromal vascular fraction in their osteoarthritis-affected joint. The final eight horses were treated the in osteoarthritis-affected joint with bone marrow-derived mesenchymal stem cells. Evaluations included clinical, radiographic, synovial fluid analysis, gross, histologic, histochemical, and biochemical evaluations. No adverse treatment-related events were observed. The model induced a significant change in all but two parameters, no significant treatment effects were demonstrated, with the exception of improvement in synovial fluid effusion PGE2 levels with bone marrow-derived mesenchymal stem cells when compared to placebo. A greater improvement was seen with bone marrow-derived mesenchymal stem cells when compared to adipose-derived stromal vascular fraction and placebo treatment. Overall, the findings of this study were not significant enough to recommend the use of stem cells for the treatment of osteoarthritis represented in this model.

  17. Repair of orbital bone defects in canines using grafts of enriched autologous bone marrow stromal cells

    PubMed Central

    2014-01-01

    Backgroud Bone tissue engineering is a new approach for the repair of orbital defects. The aim of the present study was to explore the feasibility of tissue-engineered bone constructed using bone marrow stromal cells (BMSCs) that were rapidly isolated and concentrated from bone marrow (BM) by the red cell lysis method, then combined with β-tricalcium phosphate (β-TCP) to create grafts used to restore orbital bone defects in canines. Methods In the experimental group, grafts were constructed using BMSCs obtained by red cell lysis from 20 ml bone marrow, combined with β-TCP and BM via the custom-made stem cell-scaffold device, then used to repair 10 mm diameter medial orbital wall bony defects in canines. Results were compared with those in groups grafted with BM/β-TCP or β-TCP alone, or with defects left untreated as controls. The enrichment of BMSCs and nucleated cells (NCs) in the graft was calculated from the number in untreated bone marrow and in suspensions after red cell lysis. Spiral computed tomography (CT) scans were performed 1, 4, 12 and 24 weeks after implantation in all groups. Gross examination, micro-CT and histological measurements were performed 24 weeks after surgery. The results were analyzed to evaluate the efficacy of bone repair. Results The number of NCs and of colony-forming units within the scaffolds were increased 54.8 times and 53.4 times, respectively, compared with untreated bone marrow. In the BMSC-BM/β-TCP group, CT examination revealed that the scaffolds were gradually absorbed and the bony defects were restored. Micro-CT and histological examination confirmed that the implantations led to good repair of the defects, with 6 out 8 orbital defects completely restored in the experimental group, while by contrast, the grafts in the control groups did not fully repair the bony defects, a difference which was statistically significant (p < 0.05). Conclusions Tissue-engineered bone, constructed using BMSCs isolated by red cell

  18. Manipulation of human early T lymphopoiesis by coculture on human bone marrow stromal cells: potential utility for adoptive immunotherapy.

    PubMed

    Liu, Bing; Ohishi, Kohshi; Orito, Yuki; Nakamori, Yoshiki; Nishikawa, Hiroyoshi; Ino, Kazuko; Suzuki, Kei; Matsumoto, Takeshi; Masuya, Masahiro; Hamada, Hirofumi; Mineno, Junichi; Ono, Ryoichi; Nosaka, Tetsuya; Shiku, Hiroshi; Katayama, Naoyuki

    2013-04-01

    T cell precursors are an attractive target for adoptive immunotherapy. We examined the regulation of human early T lymphopoiesis by human bone marrow stromal cells to explore in vitro manipulation of human T cell precursors in a human-only coculture system. The generation of CD7(+)CD56(-)cyCD3(-) proT cells from human hematopoietic progenitors on telomerized human bone marrow stromal cells was enhanced by stem cell factor, flt3 ligand, and thrombopoietin, but these stimulatory effects were suppressed by interleukin 3. Expression of Notch ligands Delta-1 and -4 on stromal cells additively promoted T cell differentiation into the CD7(+)cyCD3(+) pre-T cell stage, while cell growth was strongly inhibited. By combining these coculture systems, we found that initial coculture with telomerized stromal cells in the presence of stem cell factor, flt3 ligand, and thrombopoietin, followed by coculture on Delta-1- and -4-coexpressing stromal cells led to a higher percentage and number of pre-T cells. Adoptive immunotherapy using peripheral blood T cells transduced with a tumor antigen-specific T cell receptor (TCR) is a promising strategy but has several limitations, such as the risk of forming a chimeric TCR with the endogenous TCR. We demonstrated that incubation of TCR-transduced hematopoietic progenitors with the combination of coculture systems gave rise to CD7(+)TCR(+)CD3(+)CD1a(-) T cell precursors that rapidly proliferated and differentiated under the culture condition to induce mature T cell differentiation. These data show the regulatory mechanism of early T lymphopoiesis on human stromal cells and the potential utility of engineered human stromal cells to manipulate early T cell development for clinical application.

  19. Radiologic differences between bone marrow stromal and hematopoietic progenitor cell lines from Fanconi Anemia (Fancd2(-/-)) mice.

    PubMed

    Berhane, Hebist; Epperly, Michael W; Goff, Julie; Kalash, Ronny; Cao, Shaonan; Franicola, Darcy; Zhang, Xichen; Shields, Donna; Houghton, Frank; Wang, Hong; Wipf, Peter; Parmar, Kalindi; Greenberger, Joel S

    2014-01-01

    FancD2 plays a central role in the human Fanconi anemia DNA damage response (DDR) pathway. Fancd2(-/-) mice exhibit many features of human Fanconi anemia including cellular DNA repair defects. Whether the DNA repair defect in Fancd2(-/-) mice results in radiologic changes in all cell lineages is unknown. We measured stress of hematopoiesis in long-term marrow cultures and radiosensitivity in clonogenic survival curves, as well as comet tail intensity, total antioxidant stores and radiation-induced gene expression in hematopoietic progenitor compared to bone marrow stromal cell lines. We further evaluated radioprotection by a mitochondrial-targeted antioxidant GS-nitroxide, JP4-039. Hematopoiesis longevity in Fancd2(-/-) mouse long-term marrow cultures was diminished and bone marrow stromal cell lines were radiosensitive compared to Fancd2(+/+) stromal cells (Fancd2(-/-) D0 = 1.4 ± 0.1 Gy, ñ = 5.0 ± 0.6 vs. Fancd2(+/+) D0 = 1.6 ± 0.1 Gy, ñ = 6.7 ± 1.6), P = 0.0124 for D0 and P = 0.0023 for ñ, respectively). In contrast, Fancd2(-/-) IL-3-dependent hematopoietic progenitor cells were radioresistant (D0 = 1.71 ± 0.04 Gy and ñ = 5.07 ± 0.52) compared to Fancd2(+/+) (D0 = 1.39 ± 0.09 Gy and ñ = 2.31 ± 0.85, P = 0.001 for D0). CFU-GM from freshly explanted Fancd2(-/-) marrow was also radioresistant. Consistent with radiosensitivity, irradiated Fancd2(-/-) stromal cells had higher DNA damage by comet tail intensity assay compared to Fancd2(+/+) cells (P < 0.0001), slower DNA damage recovery, lower baseline total antioxidant capacity, enhanced radiation-induced depletion of antioxidants, and increased CDKN1A-p21 gene transcripts and protein. Consistent with radioresistance, Fancd2(-/-) IL-3-dependent hematopoietic cells had higher baseline and post irradiation total antioxidant capacity. While, there was no detectable alteration of radiation-induced cell cycle arrest with Fancd2(-/-) stromal cells, hematopoietic progenitor cells showed reduced G2/M cell cycle

  20. Radiologic Differences between Bone Marrow Stromal and Hematopoietic Progenitor Cell Lines from Fanconi Anemia (Fancd2−/−) Mice

    PubMed Central

    Berhane, Hebist; Epperly, Michael W.; Goff, Julie; Kalash, Ronny; Cao, Shaonan; Franicola, Darcy; Zhang, Xichen; Shields, Donna; Houghton, Frank; Wang, Hong; Wipf, Peter; Parmar, Kalindi; Greenberger, Joel S.

    2014-01-01

    FancD2 plays a central role in the human Fanconi anemia DNA damage response (DDR) pathway. Fancd2−/− mice exhibit many features of human Fanconi anemia including cellular DNA repair defects. Whether the DNA repair defect in Fancd2−/− mice results in radiologic changes in all cell lineages is unknown. We measured stress of hematopoiesis in long-term marrow cultures and radiosensitivity in clonogenic survival curves, as well as comet tail intensity, total antioxidant stores and radiation-induced gene expression in hematopoietic progenitor compared to bone marrow stromal cell lines. We further evaluated radioprotection by a mitochondrial-targeted antioxidant GS-nitroxide, JP4-039. Hematopoiesis longevity in Fancd2−/− mouse long-term marrow cultures was diminished and bone marrow stromal cell lines were radiosensitive compared to Fancd2+/+ stromal cells (Fancd2−/− D0 = 1.4 ± 0.1 Gy, ñ = 5.0 ± 0.6 vs. Fancd2+/+ D0 = 1.6 ± 0.1 Gy, ñ = 6.7 ± 1.6), P = 0.0124 for D0 and P = 0.0023 for ñ, respectively). In contrast, Fancd2−/− IL-3-dependent hematopoietic progenitor cells were radioresistant (D0 = 1.71 ± 0.04 Gy and ñ = 5.07 ± 0.52) compared to Fancd2+/+ (D0 = 1.39 ± 0.09 Gy and ñ = 2.31 ± 0.85, P = 0.001 for D0). CFU-GM from freshly explanted Fancd2−/− marrow was also radioresistant. Consistent with radiosensitivity, irradiated Fancd2−/− stromal cells had higher DNA damage by comet tail intensity assay compared to Fancd2+/+ cells (P < 0.0001), slower DNA damage recovery, lower baseline total antioxidant capacity, enhanced radiation-induced depletion of antioxidants, and increased CDKN1A-p21 gene transcripts and protein. Consistent with radioresistance, Fancd2−/− IL-3-dependent hematopoietic cells had higher baseline and post irradiation total antioxidant capacity. While, there was no detectable alteration of radiation-induced cell cycle arrest with Fancd2−/− stromal cells, hematopoietic progenitor cells showed reduced G2/M

  1. Transforming growth factor-{beta} inhibits CCAAT/enhancer-binding protein expression and PPAR{gamma} activity in unloaded bone marrow stromal cells

    SciTech Connect

    Ahdjoudj, S.; Kaabeche, K.; Holy, X.; Fromigue, O.; Modrowski, D.; Zerath, E.; Marie, P.J. . E-mail: pierre.marie@larib.inserm.fr

    2005-02-01

    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-{beta}2) 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){alpha} and C/EBP{beta} {alpha} at 5 days in bone marrow stromal cells resulting in increased peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}2) transcripts at 7 days. TGF-{beta}2 administration in unloaded rats corrected the rise in C/EBP{alpha} and C/EBP{beta} transcripts induced by unloading in bone marrow stromal cells. This resulted in inhibition of PPAR{gamma}2 expression that was associated with increased Runx2 expression. Additionally, the inhibition of C/EBP{alpha} and C/EBP{beta} expression by TGF-{beta}2 was associated with increased PPAR{gamma} serine phosphorylation in bone marrow stromal cells, a mechanism that inhibits PPAR{gamma} transactivating activity. The sequential inhibitory effect of TGF-{beta}2 on C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma}2 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-{beta}2 inhibits the excessive adipogenic differentiation of bone marrow stromal cells induced by skeletal unloading by inhibiting C/EBP{alpha}, C/EBP{beta}, and PPAR{gamma} expression and activity, which provides a sequential mechanism by which TGF-{beta}2 regulates adipogenic differentiation of bone marrow stromal cells in vivo.

  2. [Marrow stromal cells cultured in N2-supplemented medium: implications on the generation of neural cells].

    PubMed

    Castillo-Díaz, L; de la Cuétara-Bernal, K; García-Varona, A Y

    Most of the culture system for in vitro maintenance and neural differentiation of marrow stromal cells (MSCs) use synthetic media supplemented with 10 or 20% fetal bovine serum (FBS). Serum, however, is comprised of unknown quantities of undefined substances which could interfere the effect of exogenous substances on neural differentiation of MSCs. AIM. Here we describe survival of MSCs cultured in culture conditions where serum was reduced at 0.5 and 1% using Bottenstein and Sato's N2 formula (1979) and poly-L-lysine (PLL)-coated substrate. Stromal cells isolated from rat femurs were cultivated in Dulbecco's modified Eagle medium at 10, 1, 0.5% FBS or in serum free medium containing N2 formula. In serum free medium or at low serum concentration culture surface was coated with PLL. Cell survival was determined by MTT method or by counting viable cells. Survival of MSCs cultured in N2 supplement was reduced at about 40% of that observed in 10% FBS containing medium. Under these conditions cell morphology was also affected. When N2 containing medium was supplemented with FBS at 0.5 or 1% a significant increase of survival with respect to that observed in N2-supplemented cultures was observed. Cells seeded on PLL-coated surface increased their survival by contrast with their homologous cultures seeded on uncoated surface. The culture system which combines N2 formula with FBS 1% and PLL-coated surface is useful for the maintenance of MSCs. These conditions offer advantages for the study of differentiation of these cells because they reduce the confounding influence of serum. The possible implication of this culture system for the study of neural differentiation by these cells is discussed.

  3. TRPC6 regulates cell cycle progression by modulating membrane potential in bone marrow stromal cells

    PubMed Central

    Ichikawa, Jun; Inoue, Ryuji

    2014-01-01

    Background and Purpose Ca2+ influx is important for cell cycle progression, but the mechanisms involved seem to vary. We investigated the potential roles of transient receptor potential (TRP) channels and store-operated Ca2+ entry (SOCE)-related molecules STIM (stromal interaction molecule)/Orai in the cell cycle progression of rat bone marrow stromal cells (BMSCs), a reliable therapeutic resource for regenerative medicine. Experimental Approach PCR and immunoblot analyses were used to examine mRNA and protein levels, fluorescence imaging and patch clamping for Ca2+ influx and membrane potential measurements, and flow cytometry for cell cycle analysis. Key Results Cell cycle synchronization of BMSCs revealed S phase-specific enhancement of TRPC1, STIM and Orai mRNA and protein expression. In contrast, TRPC6 expression decreased in the S phase and increased in the G1 phase. Resting membrane potential (RMP) of BMSCs was most negative and positive in the S and G1 phases, respectively, and was accompanied by an enhancement and attenuation of SOCE respectively. Chemically depolarizing/hyperpolarizing the membrane erased these differences in SOCE magnitude during the cell cycle. siRNA knockdown of TRPC6 produced a negative shift in RMP, increased SOCE and caused redistribution of BMSCs with increased populations in the S and G2/M phases and accumulation of cyclins A2 and B1. A low concentration of Gd3+ (1 μM) suppressed BMSC proliferation at its concentration to inhibit SOC channels relatively specifically. Conclusions and Implications TRPC6, by changing the membrane potential, plays a pivotal role in controlling the SOCE magnitude, which is critical for cell cycle progression of BMSCs. This finding provides a new therapeutic strategy for regulating BMSC proliferation. PMID:25041367

  4. Effects of allogeneic bone marrow derived mesenchymal stromal cell therapy on voiding function in a rat model of Parkinson disease.

    PubMed

    Campeau, Lysanne; Soler, Roberto; Sittadjody, Sivanandane; Pareta, Rajesh; Nomiya, Masanori; Zarifpour, Mona; Opara, Emmanuel C; Yoo, James J; Andersson, Karl-Erik

    2014-03-01

    Cellular therapy induced transient urodynamic improvement in a rat model of Parkinson disease in which bladder dysfunction was noted after unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. We sought to prolong the effect by injecting allogeneic rat bone marrow mesenchymal stromal cells before and after microencapsulation into the substantia nigra pars compacta. Female rats underwent unilateral stereotactic injection of 6-hydroxydopamine in the medial forebrain bundle. Injection was performed in the ipsilateral substantia nigra pars compacta using vehicle alone or vehicle with nonmicroencapsulated or microencapsulated rat bone marrow derived mesenchymal stromal cells. Rats were evaluated by cystometry 7, 14, 28 and 42 days after treatment. Brains were extracted for immunostaining. At 42 days the nonmicroencapsulated group had lower threshold and intermicturition pressure, spontaneous activity and AUC than vehicle treated animals. Rats that received microencapsulated cells had lower threshold pressure at 28 days and lower spontaneous activity at 42 days than vehicle treated rats. Microencapsulated and nonmicroencapsulated rat bone marrow derived mesenchymal stromal cells were noted in the substantia nigra pars compacta up to 42 days after transplantation. At 42 days tyrosine hydroxylase positive neurons were more numerous in the substantia nigra pars compacta of the nonmicroencapsulated group, followed by the microencapsulated and vehicle treated groups. Urodynamic effects of the 6-hydroxydopamine lesion persisted up to 42 days after vehicle injection. Transplantation of nonmicroencapsulated rat bone marrow derived mesenchymal stromal cells improved urodynamic pressure by 42 days after treatment more markedly than microencapsulated cells. This was associated with more tyrosine hydroxylase positive neurons in the treated substantia nigra pars compacta of the nonmicroencapsulated group, suggesting that functional improvement requires a

  5. Reactive Oxygen Species Limit the Ability of Bone Marrow Stromal Cells to Support Hematopoietic Reconstitution in Aging Mice

    PubMed Central

    Khatri, Rahul; Krishnan, Shyam; Roy, Sushmita; Chattopadhyay, Saborni; Kumar, Vikash

    2016-01-01

    Aging of organ and abnormal tissue regeneration are recurrent problems in physiological and pathophysiological conditions. This is most crucial in case of high-turnover tissues, like bone marrow (BM). Using reciprocal transplantation experiments in mouse, we have shown that self-renewal potential of hematopoietic stem and progenitor cells (HSPCs) and BM cellularity are markedly influenced with the age of the recipient mice rather than donor mice. Moreover, accumulation of excessive reactive oxygen species (ROS) in BM stromal cells compared to HSPC compartment, in time-dependent manner, suggests that oxidative stress is involved in suppression of BM cellularity by affecting microenvironment in aged mice. Treatment of these mice with a polyphenolic antioxidant curcumin is found to partially quench ROS, thereby rescues stromal cells from oxidative stress-dependent cellular injury. This rejuvenation of stromal cells significantly improves hematopoietic reconstitution in 18-month-old mice compared to age control mice. In conclusion, this study implicates the role of ROS in perturbation of stromal cell function upon aging, which in turn affects BM's reconstitution ability in aged mice. Thus, a rejuvenation therapy using curcumin, before HSPC transplantation, is found to be an efficient strategy for successful marrow reconstitution in older mice. PMID:27140293

  6. Reactive Oxygen Species Limit the Ability of Bone Marrow Stromal Cells to Support Hematopoietic Reconstitution in Aging Mice.

    PubMed

    Khatri, Rahul; Krishnan, Shyam; Roy, Sushmita; Chattopadhyay, Saborni; Kumar, Vikash; Mukhopadhyay, Asok

    2016-06-15

    Aging of organ and abnormal tissue regeneration are recurrent problems in physiological and pathophysiological conditions. This is most crucial in case of high-turnover tissues, like bone marrow (BM). Using reciprocal transplantation experiments in mouse, we have shown that self-renewal potential of hematopoietic stem and progenitor cells (HSPCs) and BM cellularity are markedly influenced with the age of the recipient mice rather than donor mice. Moreover, accumulation of excessive reactive oxygen species (ROS) in BM stromal cells compared to HSPC compartment, in time-dependent manner, suggests that oxidative stress is involved in suppression of BM cellularity by affecting microenvironment in aged mice. Treatment of these mice with a polyphenolic antioxidant curcumin is found to partially quench ROS, thereby rescues stromal cells from oxidative stress-dependent cellular injury. This rejuvenation of stromal cells significantly improves hematopoietic reconstitution in 18-month-old mice compared to age control mice. In conclusion, this study implicates the role of ROS in perturbation of stromal cell function upon aging, which in turn affects BM's reconstitution ability in aged mice. Thus, a rejuvenation therapy using curcumin, before HSPC transplantation, is found to be an efficient strategy for successful marrow reconstitution in older mice.

  7. Cyclic mechanical stretching promotes migration but inhibits invasion of rat bone marrow stromal cells.

    PubMed

    Zhang, Bingyu; Luo, Qing; Chen, Zhe; Sun, Jinghui; Xu, Baiyao; Ju, Yang; Song, Guanbin

    2015-03-01

    Bone marrow stromal cells (BMSCs, also broadly known as bone marrow-derived mesenchymal stem cells) are multipotent stem cells that have a self-renewal capacity and multilineage differentiation potential. Mechanical stretching plays a vital role in regulating the proliferation and differentiation of BMSCs. However, little is known about the effects of cyclic stretching on BMSC migration and invasion. In this study, using a custom-made cell-stretching device, we studied the effects of cyclic mechanical stretching on rat BMSC migration and invasion using a Transwell Boyden Chamber. The protein secretion of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) was detected by gelatin zymography, and the activation of focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2) was measured by western blot. We found that cyclic mechanical stretching with 10% amplitude at 1Hz frequency for 8h promotes BMSC migration, but reduces BMSC invasion. FAK and ERK1/2 signals were activated in BMSCs after exposure to cyclic stretching. In the presence of the FAK phosphorylation blocker PF573228 or the ERK1/2 phosphorylation blocker PD98059, the cyclic-stretch-promoted migration of BMSCs was completely suppressed. On the other hand, cyclic mechanical stretching reduced the secretion of MMP-2 and MMP-9 in BMSCs, and PF573228 suppressed the cyclic-stretch-reduced secretion of MMP-2 and MMP-9. The decrease of BMSC invasion induced by mechanical stretching is partially restored by PF573228 but remained unaffected by PD98059. Taken together, these data show that cyclic mechanical stretching promotes BMSC migration via the FAK-ERK1/2 signalling pathway, but reduces BMSC invasion by decreasing secretion of MMP-2 and MMP-9 via FAK, independent of the ERK1/2 signal.

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

    PubMed

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

    2013-11-01

    The Quantum® Cell Expansion System (Quantum; Terumo BCT, Inc, Lakewood, CO, USA) is a novel hollow fiber-based device that automates and closes the cell culture process, reducing labor intensive tasks such as manual cell culture feeding and harvesting. The manual cell selection and expansion processes for the production of clinical-scale quantities of bone marrow-derived human mesenchymal stromal cells (BM-hMSCs) have been successfully translated onto the Quantum platform previously. The formerly static, manual, in vitro process performed primarily on tissue culture polystyrene substrates may raise the question of whether BM-hMSCs cultured on a hollow fiber platform yields comparable cell quality. A rigorous battery of assays was used to determine the genetic stability of BM-hMSCs selected and produced with the Quantum. In this study, genetic stability was determined by assessing spectral karyotype, micronucleus formation and tumorigenicity to resolve chromosomal aberrations in the stem cell population. Cell phenotype, adherent growth kinetics and tri-lineage differentiation were also evaluated. HMSC bone marrow aspirates, obtained from three approved donors, were expanded in parallel using T225 culture flasks and the Quantum. BM-hMSCs harvested from the Quantum demonstrated immunophenotype, morphology and tri-lineage differentiation capacity characteristics consistent with the International Society of Cell Therapy standard for hMSCs. Cell populations showed no malignant neoplastic formation in athymic mice 60 days post-transplant, no clonal chromosomal aberrations were observed and no DNA damage was found as measured by micronucleus formation. Quantum-produced BM-hMSCs are of comparable quality and demonstrate analogous genetic stability to BM-hMSCs cultured on tissue culture polystyrene substrates. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

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

    PubMed Central

    Dias, Rhayra B.; Fortuna-Costa, Anneliese; Chicaybam, Leonardo; Lopes, Daiana V.; Dutra, Hélio S.; Borojevic, Radovan; Bonamino, Martin; Mermelstein, Claudia

    2016-01-01

    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. PMID:28053606

  10. Tantalum coating of porous carbon scaffold supplemented with autologous bone marrow stromal stem cells for bone regeneration in vitro and in vivo

    PubMed Central

    Wei, Xiaowei; Wang, Benjie; Wang, Wei; Kang, Kai; Xie, Hui; Liu, Baoyi; Zhang, Xiuzhi; Zhang, Jinsong; Yang, Zhenming

    2016-01-01

    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. PMID:26843518

  11. Effects of wollastonite on proliferation and differentiation of human bone marrow-derived stromal cells in PHBV/wollastonite composite scaffolds.

    PubMed

    Li, Haiyan; Zhai, Wanying; Chang, Jiang

    2009-09-01

    In this study, the effects of wollastonite on proliferation and differentiation of human bone marrow-derived stromal cells (hBMSCs) have been investigated based on a polyhydroxybutyrate-co-hydroxyvalerate (PHBV)/ wollastonite (W) composite scaffolds system. Cell morphology, proliferation, and differentiation were measured. The results showed that the incorporation of wollastonite benefited hBMSCs adhesion, proliferation, and differentiation rate. In addition, an increase of proliferation and differentiation rate was observed when the wollastonite content in the PHBV/W composite scaffolds increased from 10 to 20 wt%. Based on our previous studies on PHBV/W composite discs, the differentiation measurements in this paper further proved that the wollastonite itself can stimulate the hBMSCs to differentiate toward osteoblasts without any osteogenic medium, and the ionic products (Ca and Si) released from wollastonite might contribute to this advantage. It is also suggested that the osteogenic differentiation of the hBMSCs can be affected by adjusting the wollastonite content in the composite scaffolds.

  12. Tantalum coating of porous carbon scaffold supplemented with autologous bone marrow stromal stem cells for bone regeneration in vitro and in vivo.

    PubMed

    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.

  13. Inferior ectopic bone formation of mesenchymal stromal cells from adipose tissue compared to bone marrow: rescue by chondrogenic pre-induction.

    PubMed

    Brocher, J; Janicki, P; Voltz, P; Seebach, E; Neumann, E; Mueller-Ladner, U; Richter, W

    2013-11-01

    Human mesenchymal stromal cells derived from bone marrow (BMSC) and adipose tissue (ATSC) represent a valuable source of progenitor cells for cell therapy and tissue engineering. While ectopic bone formation is a standard activity of human BMSC on calcium phosphate ceramics, the bone formation capacity of human ATSC has so far been unclear. The objectives of this study were to assess the therapeutic potency of ATSC for bone formation in an ectopic mouse model and determine molecular differences by standardized comparison with BMSC. Although ATSC contained less CD146(+) cells, exhibited better proliferation and displayed similar alkaline phosphatase activity upon osteogenic in vitro differentiation, cells did not develop into bone-depositing osteoblasts on β-TCP after 8weeks in vivo. Additionally, ATSC expressed less BMP-2, BMP-4, VEGF, angiopoietin and IL-6 and more adiponectin mRNA, altogether suggesting insufficient osteochondral commitment and reduced proangiogenic activity. Chondrogenic pre-induction of ATSC/β-TCP constructs with TGF-β and BMP-6 initiated ectopic bone formation in >75% of samples. Both chondrogenic pre-induction and the osteoconductive microenvironment of β-TCP were necessary for ectopic bone formation by ATSC pointing towards a need for inductive conditions/biomaterials to make this more easily accessible cell source attractive for future applications in bone regeneration.

  14. IL-6 Contributes to the Defective Osteogenesis of Bone Marrow Stromal Cells from the Vertebral Body of the Glucocorticoid-Induced Osteoporotic Mouse

    PubMed Central

    Zhang, Yuan-yuan; Yang, Hui-lin

    2016-01-01

    Osteoporosis is one of the most prevalent skeletal system diseases. It is characterized by a decrease in bone mass and microarchitectural changes in bone tissue that lead to an attenuation of bone resistance and susceptibility to fracture. Vertebral fracture is by far the most prevalent osteoporotic fracture. In the musculoskeletal system, osteoblasts, originated from bone marrow stromal cells (BMSC), are responsible for osteoid synthesis and mineralization. In osteoporosis, BMSC osteogenic differentiation is defective. However, to date, what leads to the defective BMSC osteogenesis in osteoporosis remains an open question. In the current study, we made attempts to answer this question. A mouse model of glucocorticoid-induced osteoporosis (GIO) was established and BMSC were isolated from vertebral body. The impairment of osteogenesis was observed in BMSC of osteoporotic vertebral body. The expression profiles of thirty-six factors, which play important roles in bone metabolisms, were compared through antibody array between normal and osteoporotic BMSC. Significantly higher secretion level of IL-6 was observed in osteoporotic BMSCs compared with normal control. We provided evidences that IL-6 over-secretion impaired osteogenesis of osteoporotic BMSC. Further, it was observed that β-catenin activity was inhibited in response to IL-6 over-secretion. More importantly, in vivo administration of IL-6 neutralizing antibody was found to be helpful to rescue the osteoporotic phenotype of mouse vertebral body. Our study provides a deeper insight into the pathophysiology of osteoporosis and identifies IL-6 as a promising target for osteoporosis therapy. PMID:27128729

  15. Mesenchymal stromal cells, colony-forming unit fibroblasts, from bone marrow of untreated advanced breast and lung cancer patients suppress fibroblast colony formation from healthy marrow.

    PubMed

    Hofer, Erica Leonor; Labovsky, Vivian; La Russa, Vincent; Vallone, Valeria Fernández; Honegger, Alba Elizabeth; Belloc, Carlos Gabriel; Wen, Huei Chi; Bordenave, Raúl Horacio; Bullorsky, Eduardo Oscar; Feldman, Leonardo; Chasseing, Norma Alejandra

    2010-03-01

    We have shown that bone marrow (BM) from untreated advanced lung and breast cancer patients (LCP and BCP) have a reduced number of colony-forming unit fibroblasts (CFU-Fs) or mesenchymal stem cells (MSCs). Factors that regulate the proliferation and differentiation of CFU-F are produced by the patients' BM microenvironment. We have now examined whether conditioned media (CM) from patients' CFU-F-derived stromal cells also inhibits the colony-forming efficiency (CFE) of CFU-F in primary cultures from healthy volunteers (HV)-BM. Thus the number and proliferation potential of HV-CFU-F were also found to be decreased and similar to colony numbers and colony size of patients' CFU-F. Stromal cells from both of these types of colonies appeared relatively larger and lacked the characteristic spindle morphology typically seen in healthy stromal cells. We developed an arbitrary mesenchymal stromal cell maturational index by taking three measures consisting of stromal cell surface area, longitudinal and horizontal axis. All stromal indices derived from HV-CFU-F grown in patients' CM were similar to those from stromal elements derived from patients' CFU-F. These indices were markedly higher than stromal indices typical of HV-CFU-F cultured in healthy CM or standard medium [alpha-medium plus 20% heat-inactivated fetal bovine serum (FBS)]. Patients' CM had increased concentrations of the CFU-F inhibitor, GM-CSF, and low levels of bFGF and Dkk-1, strong promoters of self-renewal of MSCs, compared to the levels quantified in CM from HV-CFU-F. Moreover, the majority of patients' MSCs were unresponsive in standard medium and healthy CM to give CFU-F, indicating that the majority of mesenchymal stromal cells from patients' CFU-F are locked in maturational arrest. These results show that alterations of GM-CSF, bFGF, and Dkk-1 are associated with deficient cloning and maturation arrest of CFU-F. Defective autocrine and paracrine mechanisms may be involved in the BM microenvironments of

  16. A method for measurement of drug sensitivity of myeloma cells co-cultured with bone marrow stromal cells.

    PubMed

    Misund, Kristine; Baranowska, Katarzyna A; Holien, Toril; Rampa, Christoph; Klein, Dionne C G; Børset, Magne; Waage, Anders; Sundan, Anders

    2013-07-01

    The tumor microenvironment can profoundly affect tumor cell survival as well as alter antitumor drug activity. However, conventional anticancer drug screening typically is performed in the absence of stromal cells. Here, we analyzed survival of myeloma cells co-cultured with bone marrow stromal cells (BMSC) using an automated fluorescence microscope platform, ScanR. By staining the cell nuclei with DRAQ5, we could distinguish between BMSC and myeloma cells, based on their staining intensity and nuclear shape. Using the apoptotic marker YO-PRO-1, the effects of drug treatment on the viability of the myeloma cells in the presence of stromal cells could be measured. The method does not require cell staining before incubation with drugs, and less than 5000 cells are required per condition. The method can be used for large-scale screening of anticancer drugs on primary myeloma cells. This study shows the importance of stromal cell support for primary myeloma cell survival in vitro, as half of the cell samples had a marked increase in their viability when cultured in the presence of BMSC. Stromal cell-induced protection against common myeloma drugs is also observed with this method.

  17. Bone marrow stromal cells inhibits HMGB1-mediated inflammation after stroke in type 2 diabetic rats.

    PubMed

    Hu, J; Liu, B; Zhao, Q; Jin, P; Hua, F; Zhang, Z; Liu, Y; Zan, K; Cui, G; Ye, X

    2016-06-02

    High-mobility group box 1 (HMGB1), a ligand of receptor for advanced glycation endproducts (RAGE), functions as a proinflammatory factor. It is mainly involved in inflammatory activation and contributes to the initiation and progression of stroke. By using a model of transient middle cerebral artery occlusion (MCAo) in type 2 diabetic rats, we investigated the changes of pro-inflammation mediators, blood-brain barrier (BBB) leakage and functional outcome after stroke. Type 2 diabetic rats did not show an increased lesion volume, but exhibited significantly increased expression of HMGB1 and RAGE, BBB leakage, as well as decreased functional outcome after stroke compared with control rats. Injection of bone marrow stromal cells (BMSCs) into type 2 diabetic rats significantly reduced the expression of HMGB1 and RAGE, attenuated BBB leakage, and improved functional outcome after stroke. BMSCs-treated type 2 diabetic rats inhibited inflammation and improved functional outcome after stroke. Furthermore, in vitro data support the hypothesis that BMSCs-induced reduction of HMGB1 and RAGE in T2DM-MCAo rats contributed to attenuated inflammatory response in the ischemic brain, which may lead to the beneficial effects of BMSCs treatment. Further investigation of BMSCs treatment in type 2 diabetic stroke is warranted.

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

    PubMed

    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.

  19. A crucial role of caspase-3 in osteogenic differentiation of bone marrow stromal stem cells

    PubMed Central

    Miura, Masako; Chen, Xiao-Dong; Allen, Matthew R.; Bi, Yanming; Gronthos, Stan; Seo, Byoung-Moo; Lakhani, Saquib; Flavell, Richard A.; Feng, Xin-Hua; Robey, Pamela Gehron; Young, Marian; Shi, Songtao

    2004-01-01

    Caspase-3 is a critical enzyme for apoptosis and cell survival. Here we report delayed ossification and decreased bone mineral density in caspase-3–deficient (Casp3–/– and Casp3+/–) mice due to an attenuated osteogenic differentiation of bone marrow stromal stem cells (BMSSCs). The mechanism involved in the impaired differentiation of BMSSCs is due, at least partially, to the overactivated TGF-β/Smad2 signaling pathway and the upregulated expressions of p53 and p21 along with the downregulated expressions of Cdk2 and Cdc2, and ultimately increased replicative senescence. In addition, the overactivated TGF-β/Smad2 signaling may result in the compromised Runx2/Cbfa1 expression in preosteoblasts. Furthermore, we demonstrate that caspase-3 inhibitor, a potential agent for clinical treatment of human diseases, caused accelerated bone loss in ovariectomized mice, which is also associated with the overactivated TGF-β/Smad2 signaling in BMSSCs. This study demonstrates that caspase-3 is crucial for the differentiation of BMSSCs by influencing TGF-β/Smad2 pathway and cell cycle progression. PMID:15599395

  20. Nanotopography Induced Human Bone Marrow Mesangiogenic Progenitor Cells (MPCs) to Mesenchymal Stromal Cells (MSCs) Transition

    PubMed Central

    Antonini, Sara; Montali, Marina; Jacchetti, Emanuela; Meucci, Sandro; Parchi, Paolo D.; Barachini, Serena; Panvini, Francesca M.; Pacini, Simone; Petrini, Iacopo; Cecchini, Marco

    2016-01-01

    Mesangiogenic progenitor cells (MPCs) are a very peculiar population of cells present in the human adult bone marrow, only recently discovered and characterized. Owing to their differentiation potential, MPCs can be considered progenitors for mesenchymal stromal cells (MSCs), and for this reason they potentially represent a promising cell population to apply for skeletal tissue regeneration applications. Here, we evaluate the effects of surface nanotopography on MPCs, considering the possibility that this specific physical stimulus alone can trigger MPC differentiation toward the mesenchymal lineage. In particular, we exploit nanogratings to deliver a mechanical, directional stimulus by contact interaction to promote cell morphological polarization and stretching. Following this interaction, we study the MPC-MSC transition by i. analyzing the change in cell morphotype by immunostaining of the key cell-adhesion structures and confocal fluorescence microscopy, and ii. quantifying the expression of cell-phenotype characterizing markers by flow cytometry. We demonstrate that the MPC mesengenic differentiation can be induced by the solely interaction with the NGs, in absence of any other external, chemical stimulus. This aspect is of particular interest in the case of multipotent progenitors as MPCs that, retaining both mesengenic and angiogenic potential, possess a high clinical appeal. PMID:28066765

  1. Sulfated hyaluronan alters fibronectin matrix assembly and promotes osteogenic differentiation of human bone marrow stromal cells

    NASA Astrophysics Data System (ADS)

    Vogel, Sarah; Arnoldini, Simon; Möller, Stephanie; Schnabelrauch, Matthias; Hempel, Ute

    2016-11-01

    Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC.

  2. Effects of plating density and culture time on bone marrow stromal cell characteristics.

    PubMed

    Neuhuber, Birgit; Swanger, Sharon A; Howard, Linda; Mackay, Alastair; Fischer, Itzhak

    2008-09-01

    Bone marrow stromal cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in disorders including cardiac infarction, stroke, and spinal cord injury. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of initial plating density and total time in culture on proliferation, cell morphology, and differentiation potential of heterogeneous MSC cultures and more homogeneous cloned subpopulations. Rat MSC were plated at 20, 200, and 2000 cells/cm(2) and grown to 50% confluency. The numbers of population doublings and doubling times were determined within and across multiple passages. Changes in cell morphology and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages were evaluated and compared among early, intermediate, and late passages, as well as between heterogeneous and cloned MSC populations. We found optimal cell growth at a plating density of 200 cells/cm(2). Cultures derived from all plating densities developed increased proportions of flat cells over time. Assays for chondrogenesis, osteogenesis, and adipogenesis showed that heterogeneous MSC plated at all densities sustained the potential for all three mesenchymal phenotypes through at least passage 5; the flat subpopulation lost adipogenic and chondrogenic potential. Our findings suggest that the initial plating density is not critical for maintaining a well-defined, multipotent MSC population. Time in culture, however, affects cell characteristics, suggesting that cell expansion should be limited, especially until the specific characteristics of different MSC subpopulations are better understood.

  3. Cerium oxide nanoparticles protect primary mouse bone marrow stromal cells from apoptosis induced by oxidative stress

    NASA Astrophysics Data System (ADS)

    Zhang, Qun; Ge, Kun; Duan, Jianlei; Chen, Shizhu; Zhang, Ran; Zhang, Cuimiao; Wang, Shuxiang; Zhang, Jinchao

    2014-11-01

    Cerium oxide nanoparticles (nanoceria) have been widely used in industries and biomedical fields due to its unique properties. Previous biodistribution studies of nanoceria in vivo have shown that they are accumulated in the bone of mice after intravenous administration, about 20 % of the total intake, however, the potential effect and the mechanism of nanoceria on bone metabolism are not well-understood. Our results showed that both 25 and 50 nm nanceria decreased the damage of cell viability induced by H2O2 in a dose-dependent manner. The apoptosis ratio of pre-incubated group with nanoceria was lower than the H2O2 group. The cellular uptake studies indicated that there was a dose-dependent accumulation of both two size nanoparticles in bone marrow stromal cells. Nanoceria could be uptaken by cells due to the synergistic effect of multiple endocytosis mechanisms, and then evenly distributed in the cytoplasm without entering the nucleus. Our results suggest that nanoceria could reduce intracellular ROS level induced by H2O2 in a dose-dependent manner, moreover, maintain the normal function of mitochondria, suggesting nanoceria may have potent applications for preventing or treating osteoporosis.

  4. Voltage-dependent calcium and chloride currents in S17 bone marrow stromal cell line.

    PubMed

    Silva, Henrique B; Medei, Emiliano; Rodrigues, Deivid C; Rondinelli, Edson; Almeida, Norma A S; Goldenberg, Regina C S; de Carvalho, Antonio C Campos; Nascimento, José H M

    2010-04-01

    The bone marrow stromal cell line S17 has been used to study hematopoiesis in vitro. In this study, we demonstrate the presence of calcium and chloride currents in cultured S17 cells. Calcium currents were of low amplitude or barely detectable (50-100 pA). Hence to amplify the currents, we have used barium as a charge carrier. Barium currents were identified based on their distinct voltage-dependence, and sensitivity to dihydropyridines. S17 cells also exhibited a slowly activating outward current without inactivation, most commonly seen when the sodium of the extracellular solution was replaced either by TEA (TEA/Cs saline) or NMDG (NMDG saline), or by addition of amiloride to the extracellular solution. This current was abolished either by 500 microM SITS (4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid) or 500 microM DPC (diphenylamine-2-carboxylic acid) a cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel blocker, identifying it as a Cl(-) current. RT-PCR identified the presence of ENaC and CFTR transcripts. CFTR blockade reduced cell proliferation, suggesting that this channel plays a physiological role in regulation of S17 cell proliferation.

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

    PubMed

    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.

  6. Phenotypic characteristics of hybrid cells generated by transferring neuronal nuclei into bone marrow stromal cell cytoplasts.

    PubMed

    Zhou, Zhujuan; Xu, Yan; Zhong, Qi; Zheng, Jian

    2012-02-10

    Bone marrow stromal cells (BMSCs) are promising donor cells for transplantation therapies for a variety of diseases. However, there still lack efficient ways to induce directional differentiation of BMSCs to promote their practical use in transplantation therapy. In this study, we constructed hybrid cells by transferring neuronal nuclei into BMSC cytoplasts and investigated the proliferative capacity and phenotypic characteristics of the hybrid cells. The neuronal nuclei were labeled with Hoechst 33342 before the transfer process, and the cell membrane antigen CD71 was used as a marker of BMSC cytoplasts. The BMSC cytoplasts and neuronal karyoplasts were separated by Ficoll density gradient ultracentrifugation. The hybrid cells were generated by the polyethylene glycol-mediated fusion of BMSC cytoplasts with neuronal karyoplasts. The hybrid cells exhibited Hoechst 33342 staining in their nuclei and CD71 staining on their cytomembranes, which confirmed the success of cell fusion. The hybrid cells were positive for BrdU immunostaining. Viability analysis of the cultured hybrid cells by the MTT assay demonstrated their proliferative ability. Immunocytochemical staining revealed the expression of the neuron-specific markers NeuN and MAP2 in the third passage hybrid cells, which indicated their neuronal phenotypic characteristics. The results demonstrated that the hybrid cells produced by fusing neuronal karyoplasts with BMSC cytoplasts had proliferative capability and expressed the neuron-specific markers. Further study is required to investigate the phenotype of the hybrid cells both structurally and functionally.

  7. Grafted bone marrow stromal cells: a contributor to glial repair after spinal cord injury.

    PubMed

    Zhang, Li-Xin; Yin, Yan-Mei; Zhang, Zhi-Qiang; Deng, Ling-Xiao

    2015-06-01

    In the CNS, astrocytes, oligodendrocytes and microglias are involved in not only development but also pathology such as spinal cord injury (SCI). Glial cells play dual roles (negative vs. positive effects) in these processes. After SCI, detrimental effects usually dominate and significantly retard functional recovery, and curbing these effects is critical for promoting neurological improvement. Bone marrow stromal cells (BMSCs) represent a new therapeutic approach for SCI by enabling improved sensory and motor functions in animal models. Although transdifferentiation to spinal neurons was poor, because of their pleiotropic nature, the protective effects of BMSCs are broad and are primarily mediated through modulation of transdifferentiation into host spinal glial components. Transplantation of BMSCs can positively alter the spinal microenvironment and enhance recovery. The objective of this review is to discuss these and other related mechanisms. Since BMSCs transplantation has been applied in other clinical fields, we hope to provide useful clues for the clinical application of BMSCs to treat the SCI in the near future.

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

    Ren Hongying; Cai Huiguo; Han Zhongchao; Yang Renchi; Zhao, Qinjun; Cao Ying; Li Jing; Zhou Cixiang; Liao Lianming; Jia Mingyue; Zhao Qian; Chen Guoqiang . E-mail: chengq@shsmu.edu.cn; Zhao, R.C. |. 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.

  10. Identification of differentiation-inducing activity produced by human bone marrow stromal cell line LP101.

    PubMed

    Hiramoto, Masaki; Kawakami, Yutaka; Nabeshima, Ryusuke; Shima, Daisuke; Handa, Hiroshi; Aizawa, Shin

    2004-11-01

    We have previously reported that human promyelocytic leukemia HL-60 cells can be induced to differentiate into mature granulocytes when HL-60 co-cultivated with human bone marrow stromal LP101 cells. In the present study, we investigated which factors produced by LP101 cells induce HL-60 cells to differentiate into mature granulocytes. The expression of the cell surface antigen CD11b on HL-60 cells was increased after a 72-h culture with the conditioned medium (CM) obtained from LP101 cells. LP101 cells were observed to produce various cytokines, including TNF-alpha, GM-CSF and IL-6. The neutralizing antibodies against these cytokines partially suppressed the CM-induced differentiation of HL-60 cells. Recombinant TNF-alpha induced the differentiation of HL-60 cells, and GM-CSF and IL-6 additionally enhanced the effect of TNF-alpha. When the CM was divided into a low molecular weight (LMW) fraction and a high molecular weight (HMW) fraction by ultrafiltration, the LMW fraction synergistically enhanced the differentiation inducible activity of TNF-alpha. These results demonstrate that LP101 cells induce the differentiation of HL-60 cells by producing various cytokines including TNF-alpha, IL-6, and GM-CSF, and that unknown low molecular weight factors also participate.

  11. Calcium deposition in photocrosslinked poly(Pro-Hyp-Gly) hydrogels encapsulated rat bone marrow stromal cells.

    PubMed

    Nurlidar, Farah; Yamane, Keisuke; Kobayashi, Mime; Terada, Kayo; Ando, Tsuyoshi; Tanihara, Masao

    2017-07-17

    Reproducing the features of the extracellular matrix is important for fabricating three-dimensional (3D) scaffolds for tissue regeneration. A collagen-like polypeptide, poly(Pro-Hyp-Gly), is a promising material for 3D scaffolds because of its excellent physical properties, biocompatibility, and biodegradability. In this paper, we present a novel photocrosslinked poly(Pro-Hyp-Gly) hydrogel as a 3D scaffold for simultaneous rat bone marrow stromal cell (rBMSC) encapsulation. The hydrogels were fabricated using visible-light photocrosslinking at various concentrations of methacrylated poly(Pro-Hyp-Gly) (20-50 mg/mL) and irradiation times (3 or 5 min). The results show that the rBMSCs encapsulated in the hydrogels survived seven days of incubation. Calcium deposition on the encapsulated rBMSCs was assessed with SEM observation, Alizarin Red S and von Kossa staining. The most strongly stained area was observed in the hydrogel formed with 30 mg/mL of methacrylated poly(Pro-Hyp-Gly) with 5 min irradiation. These findings demonstrate that poly(Pro-Hyp-Gly) hydrogels support rBMSC viability and differentiation, as well as demonstrating the feasibility of using poly(Pro-Hyp-Gly) hydrogels as a cytocompatible, biodegradable 3D scaffold for tissue regeneration. This article is protected by copyright. All rights reserved.

  12. The interaction between bone marrow stromal cells and RGD modified three dimensional porous polycaprolactone scaffolds

    PubMed Central

    Zhang, Huina; Lin, Chia-Ying; Hollister, Scott J

    2015-01-01

    We previously established a simple method to immobilize the Arg-Gly-Asp (RGD) peptide on polycaprolactone (PCL) two-dimensional film surfaces that significantly improved bone marrow stromal cell (BMSC) adhesion to these films. The current work extends this modification strategy to three-dimensional (3D) PCL scaffolds to investigate BMSCs attachment, cellular distribution and cellularity, signal transduction and survival on the modified PCL scaffold compared to those on the untreated ones. The results demonstrated that treatment of 3D PCL scaffold surfaces with 1,6-hexanediamine introduced the amino functional groups onto the porous PCL scaffold homogenously as detected by a ninhydrin staining method. Followed by the cross-linking reaction, RGDC peptide was successfully immobilized on the surface of PCL scaffold. Although the static seeding method used in this study caused heterogeneous cell distribution, the RGD modified PCL scaffold still demonstrated the improved BMSC attachment and cellular distribution in the scaffold. More importantly, the integrin-mediated signal transduction FAK-PI3K-Akt pathway was significantly up-regulated by RGD modification and a subsequent increase in cell survival and growth was found in the modified scaffold. The present study introduces an easy method to immobilize RGD peptide on the 3D porous PCL scaffold and provides further evidence that modification of 3D PCL scaffolds with RGD peptides elicits specific cellular responses and improves the final cell-biomaterial interaction. PMID:19487019

  13. Multilayer tendon slices seeded with bone marrow stromal cells: a novel composite for tendon engineering.

    PubMed

    Omae, Hiromichi; Zhao, Chunfeng; Sun, Yu Long; An, Kai-Nan; Amadio, Peter C

    2009-07-01

    The ideal scaffold for tendon engineering would possess the basic structure of the tendon, native extracellular matrix, and capability of cell seeding. The purpose of this study was to assess the tissue engineering potential of a novel composite consisting of a decellularized multilayer sliced tendon (MST) scaffold seeded with bone marrow stromal cells (BMSC). BMSC and infraspinatus tendons were harvested from 20 dogs. The tendons were sectioned in longitudinal slices with a thickness of 50 microm. The slices were decellularized, seeded with BMSC, and then bundled into one composite. The composite was incubated in culture media for 14 days. The resulting BMSC-seeded MST was evaluated by qRT-PCR and histology. The BMSC viability was assessed by a fluorescent tracking marker. Histology showed that the seeded cells aligned between the collagen fibers of the tendon slices. Analysis by qRT-PCR showed higher tenomodulin and MMP13 expression and lower collagen type I expression in the composite than in the BMSC before seeding. BMSC labeled with fluorescent tracking marker were observed in the composite after culture. Mechanical testing showed no differences between scaffolds with or without BMSC. BMSC can survive in a MST scaffold. The increased tenomodulin expression suggests that BMSC might express a tendon phenotype in this environment. This new composite might be useful as a model of tendon tissue engineering.

  14. Sulfated hyaluronan alters fibronectin matrix assembly and promotes osteogenic differentiation of human bone marrow stromal cells

    PubMed Central

    Vogel, Sarah; Arnoldini, Simon; Möller, Stephanie; Schnabelrauch, Matthias; Hempel, Ute

    2016-01-01

    Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC. PMID:27808176

  15. [Morphological results of stromal stem cells of bone marrow origin into the thrombosed vein in experiment].

    PubMed

    Maĭborodin, I V; Morozov, V V; Novikova, Ia V; Matveeva, V A; Artem'eva, L V; Matveev, A L; Komenko, S V; Marchukov, S V

    2012-01-01

    Using the methods of luminescent microscopy, the results of injection of autologous multipotent stromal (mesenchymal) stem cells of bone marrow origin (SSCBMO) containing GFP gene, into thrombosed hindlimb vein were studied in 226 male Wag rats. It was found that the restoration of blood flow through the thrombosed main vein was not always the result of thrombolysis. No signs of incorporation of injected SSCBMO into the wall of thrombosed vessel, clot recanalization or collateral formation were detected. In experimental thrombosis model with thrombin administration and main vein ligation, the thrombosis of its small branches also took place. The restoration of blood flow occured via either blood clot recanalization or obliteration of thrombosed vessels and the outgrowth of the new ones. SSCBMO were found to participate in both of these processes resulting in faster restoration of a blood flow in the tissue microregion of thrombosed vein. Gradually the injected SSCBMO and the structures formed with their participation, were replaced by the own cells of a recipient organism.

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

    SciTech Connect

    Kramer, Brian C.; Woodbury, Dale . E-mail: WOODBURYDL@AOL.COM; Black, Ira B.

    2006-05-19

    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, GFR{alpha}1, 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.

  17. Bystander effect in glioma suicide gene therapy using bone marrow stromal cells.

    PubMed

    Li, Shaoyi; Gu, Chunyu; Gao, Yun; Amano, Shinji; Koizumi, Shinichiro; Tokuyama, Tsutomu; Namba, Hiroki

    2012-11-01

    An established rat intracranial glioma was successfully treated through the tumoricidal bystander effect generated by intratumoral injection of rat bone marrow stromal cells (BMSCs) transduced with the herpes simplex virus-thymidine kinase gene (BMSCtk cells) followed by systemic ganciclovir administration. In the present study, we tested the bystander effect of this treatment strategy when using human BMSCs as the vector cells. Human BMSCtk cells were mixed with various kinds of brain tumor cell lines (human and rat glioma cells) and examined in vitro and in vivo tumoricidal bystander effects, by co-culture study and co-implantation study in the nude mouse, respectively. A significant in vitro bystander effect was observed between human BMSCtk cells and any of the tumor cells examined in the ganciclovir-containing medium. A potent in vivo bystander effect against human and rat glioma cells was also demonstrated when ganciclovir was administered. Migratory activity of the human BMSCs toward the tumor cells was enhanced by the conditioned media obtained from both human and rat glioma cells compared to the fresh media. The results of this study have demonstrated that the bystander effect generated by BMSCtk cells and ganciclovir is not cell type-specific, suggesting that the strategy would be quite feasible for clinical use. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. The LIM protein LIMD1 influences osteoblast differentiation and function

    SciTech Connect

    Luderer, Hilary F.; Bai Shuting; Longmore, Gregory D.

    2008-09-10

    The balance between bone resorption and bone formation involves the coordinated activities of osteoblasts and osteoclasts. Communication between these two cell types is essential for maintenance of normal bone homeostasis; however, the mechanisms regulating this cross talk are not completely understood. Many factors that mediate differentiation and function of both osteoblasts and osteoclasts have been identified. The LIM protein Limd1 has been implicated in the regulation of stress osteoclastogenesis through an interaction with the p62/sequestosome protein. Here we show that Limd1 also influences osteoblast progenitor numbers, differentiation, and function. Limd1{sup -/-} calvarial osteoblasts display increased mineralization and accelerated differentiation. While no significant differences in osteoblast number or function were detected in vivo, bone marrow stromal cells isolated from Limd1{sup -/-} mice contain significantly more osteoblast progenitors compared to wild type controls when cultured ex vivo. Furthermore, we observed a significant increase in nuclear {beta}-catenin staining in differentiating Limd1{sup -/-} calvarial osteoblasts suggesting that Limd1 is a negative regulator of canonical Wnt signaling in osteoblasts. These results demonstrate that Limd1 influences not only stress osteoclastogenesis but also osteoblast function and osteoblast progenitor commitment. Together, these data identify Limd1 as a novel regulator of both bone osetoclast and bone osteoblast development and function.

  19. P-glycoprotein overexpression in bone marrow-derived multipotent stromal cells decreases the risk of steroid-induced osteonecrosis in the femoral head.

    PubMed

    Han, Ning; Li, Zengchun; Cai, Zhengdong; Yan, Zuoqin; Hua, Yingqi; Xu, Chong

    2016-11-01

    P-glycoprotein (P-gp) plays a role in steroid-induced osteonecrosis of the femoral head (ONFH), but the underlying mechanism remains unknown. We hypothesized that P-gp overexpression can prevent ONFH by regulating bone marrow-derived multipotent stromal cell (BMSC) adipogenesis and osteogenesis. BMSCs from Sprague-Dawley rats were transfected with green fluorescent protein (GFP) or the multidrug resistance gene 1 (MDR1) encoding GFP and P-gp. Dexamethasone was used to induce BMSC differentiation. Adipogenesis was determined by measuring peroxisome proliferator-activated receptor (PPAR-γ) expression and the triglyceride level. Osteogenesis was determined by measuring runt-related transcription factor 2 (Runx2) expression and alkaline phosphatase activity. For in vivo experiments, rats were injected with saline, BMSCs expressing GFP (GFP-BMSCs) or BMSCs expressing GFP-P-gp (MDR1-GFP-BMSCs). After dexamethasone induction, adipogenesis was determined by measuring PPAR-γ expression and fatty marrow, whereas osteogenesis was detected by measuring Runx2 expression, trabecular parameters and the mineral apposition rate, followed by evaluation of the incidence of ONFH. Overexpression of P-gp in BMSCs resulted in markedly decreased expression of adipogenic markers and increased expression of osteogenic markers. Compared with rats injected with saline, rats injected with GFP-BMSCs showed reduced ONFH, and the injected GFP-positive BMSCs attached to trabecular surfaces and exhibited an osteoblast-like morphology. Compared with the rats injected with BMSCs expressing GFP alone, rats injected with BMSCs overexpressing GFP and P-gp showed lower adipocytic variables, higher osteogenic variables and lower incidence of ONFH. Overexpression of P-gp inhibited BMSC adipogenesis and promoted osteogenesis, which reduced the incidence of steroid-induced ONFH. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular

  20. Kinetics of hematopoietic stem cells and supportive activities of stromal cells in a three-dimensional bone marrow culture system.

    PubMed

    Harada, Tomonori; Hirabayashi, Yukio; Hatta, Yoshihiro; Tsuboi, Isao; Glomm, Wilhelm Robert; Yasuda, Masahiro; Aizawa, Shin

    2015-01-01

    In the bone marrow, hematopoietic cells proliferate and differentiate in close association with a three-dimensional (3D) hematopoietic microenvironment. Previously, we established a 3D bone marrow culture system. In this study, we analyzed the kinetics of hematopoietic cells, and more than 50% of hematopoietic progenitor cells, including CFU-Mix, CFU-GM and BFU-E in 3D culture were in a resting (non-S) phase. Furthermore, we examined the hematopoietic supportive ability of stromal cells by measuring the expression of various mRNAs relevant to hematopoietic regulation. Over the 4 weeks of culture, the stromal cells in the 3D culture are not needlessly activated and "quietly" regulate hematopoietic cell proliferation and differentiation during the culture, resulting in the presence of resting hematopoietic stem cells in the 3D culture for a long time. Thus, the 3D culture system may be a new tool for investigating hematopoietic stem cell-stromal cell interactions in vitro.

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

    NASA Astrophysics Data System (ADS)

    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

  2. In vitro quantitation of lethal and physiologic effects of total body irradiation on stromal and hematopoietic stem cells in continuous bone marrow cultures from Rf mice

    SciTech Connect

    Greenberger, J.S.; Eckner, R.J.; Otten, J.A.; Tennant, R.W.

    1982-07-01

    The effects of in vivo total body irradiation (TBI) and interval from TBI to explant of marrow on: stromal cell proliferation in vitro; stromal cell support of hematopoiesis in continuous bone marrow culture; and generation of WEHI-3 growth factor (GF)-dependent lines of hematopoietic progenitor cells were evaluated. Explant of marrow at 2, 4, 5, or 6 months after single fraction TBI (300-800 rad) was associated with decreased longevity of hemopoiesis and a decrease in the proliferative capacity of fibroblastic adherent-stromal colony forming cells (CFUf) as measured by colony size at 14 days and number of colonies per 10/sup 6/ cells plated. In contrast, explant of marrow 8 to 24 months after TBI produced cultures with longevity that was indistinguishable from age-matched control cultures (19-24 weeks). Marrow from irradiated first and second generation recipients of serially transferred marrow demonstrated a similar 7-month in vivo recovery period; however, the plateau maximum duration of hemopoiesis did not return to control levels. Purified stromal cell cultures were prepared by corticosteroid-deprivation of explanted marrow for 28 days and were then engrafted in vitro with marrow from C57BL/6J or RfM/UN mice that had been irradiated 1 month previously. Hemopoiesis in these cultures was restored, and they produced GM-CFUc and granulocytes for 15-24 weeks. Thus, healthy stroma supported growth of recently irradiated hemopoietic cells in vitro. Indirect effects of x-irradiation on hemopoietic stem cells through damage and repair in the stromal cell compartment can be effectively studied with the present bone marrow culture system. (JMT)

  3. Hematopoiesis on cellulose ester membranes. XI. Induction of new bone and a hematopoietic microenvironment by matrix factors secreted by marrow stromal cells.

    PubMed

    Knospe, W H; Husseini, S G; Fried, W

    1989-07-01

    Cellulose ester membranes (CEM) were coated with stromal cells from bone marrow (BM) or bone and implanted intraperitoneally (IP) in CAF1 mice for intervals of 1 to 6 months. Previous studies indicated that matrix factors [glycoproteins (GPs), proteoglycans (PGs), and glycosaminoglycans (GAGs)] were secreted by the regenerating stromal cells and adsorbed by the CEM. After 1 to 6 months, the CEMs were removed, scraped free of adherent cells, and irradiated in vitro with 40 Gy. The scraped and irradiated CEMs were then reimplanted IP or subcutaneously (SC) for periods of 1 to 6 months in secondary syngeneic murine hosts. They were then removed for histologic study. CEMs reimplanted in SC sites developed bone and hematopoiesis as early as 1 month after implantation. Maximum hematopoiesis and bone formation was observed after 3 months. CEMs coated during the initial implantation with bone-derived stromal cells contained more bone and hematopoietic cells than did CEMs coated with marrow-derived stromal cells after SC implementation. Neither the CEMs coated with bone stromal cells nor those coated with marrow stromal cells developed new bone or trilineal hematopoiesis after being implanted IP. A few CEMs contained small foci of granulopoiesis only. We conclude that noncellular matrix substances deposited on CEMs by bone, and to a lesser degree by marrow cells, can induce prestromal cells in the SC tissues to produce a microenvironment suitable for trilineal hematopoiesis.

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

    PubMed

    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.

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

    SciTech Connect

    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

    2010-12-10

    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.

  6. EFFECTS OF PLATING DENSITY AND CULTURE TIME ON BONE MARROW STROMAL CELL CHARACTERISTICS

    PubMed Central

    Neuhuber, Birgit; Swanger, Sharon A.; Howard, Linda; Mackay, Alastair; Fischer, Itzhak

    2008-01-01

    Objective Bone marrow stromal cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in disorders including cardiac infarction, stroke and spinal cord injury. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of initial plating density and total time in culture on proliferation, cell morphology, and differentiation potential of heterogeneous MSC cultures and more homogeneous cloned subpopulations. Methods Rat MSC were plated at 20, 200 and 2000 cells/cm2 and grown to 50% confluency. The numbers of population doublings and doubling times were determined within and across multiple passages. Changes in cell morphology and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages were evaluated and compared among early, intermediate and late passages, as well as between heterogeneous and cloned MSC populations. Results We found optimal cell growth at a plating density of 200 cells/cm2. Cultures derived from all plating densities developed increased proportions of flat cells over time. Assays for chondrogenesis, osteogenesis and adipogenesis showed that heterogeneous MSC plated at all densities sustained the potential for all three mesenchymal phenotypes through at least passage 5; the flat subpopulation lost adipogenic and chondrogenic potential. Conclusion Our findings suggest that the initial plating density is not critical for maintaining a well-defined, multipotent MSC population. Time in culture, however, affects cell characteristics, suggesting that cell expansion should be limited, especially until the specific characteristics of different MSC subpopulations are better understood. PMID:18495329

  7. Effects of in vivo deletion of GATA2 in bone marrow stromal cells.

    PubMed

    Hasegawa, Shin; Fujiwara, Tohru; Okitsu, Yoko; Kato, Hiroki; Sato, Yuki; Fukuhara, Noriko; Onishi, Yasushi; Shimizu, Ritsuko; Yamamoto, Masayuki; Harigae, Hideo

    2017-09-01

    The bone marrow (BM) microenvironment comprises multiple stem cell niches derived from BM mesenchymal stem cells (MSCs). Previous in vitro analyses have suggested that transcription factor GATA2 plays an important role in adipocyte differentiation of BM-MSCs and in hematopoietic support, but the role of GATA2 in vivo remains unknown. We evaluated GATA2 effects in BM-MSCs in vivo. Expression profiling analysis of Gata2-knockout Ter119(-)CD45(-) mesenchymal stromal cells obtained from compact bone from tamoxifen-treated Gata2 conditional knockout mice (Gata2(f/f)/ER-Cre mice) revealed upregulation of 110 genes and downregulation of 141 genes by a factor of 2. Moreover, gene ontology analysis revealed significant enrichment of genes involved in cell adhesion and chemotaxis. We did not find any phenotypic changes when Gata2 was deleted with BM-MSC-related gene promoters, such as Nestin, Prx1, and Lepr, except for a significant decrease in the colony number of Gata2(f/f)/Prx1-Cre mice. There was a significant decrease in the percentage of the common myeloid progenitor fraction when Gata2 was deleted in all BM cells, except hematopoietic cells after normal BM cells were transplanted into irradiated Gata2(f/f)/ER-Cre mice with Gata2 subsequently knocked out by tamoxifen administration. In conclusion, GATA2 could affect the function of BM-MSCs in vivo, presumably by regulating the expression of extracellular signals. Copyright © 2017 ISEH – Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

  8. Mechanical stimulation orchestrates the osteogenic differentiation of human bone marrow stromal cells by regulating HDAC1.

    PubMed

    Wang, J; Wang, C D; Zhang, N; Tong, W X; Zhang, Y F; Shan, S Z; Zhang, X L; Li, Q F

    2016-05-12

    Mechanical stimulation and histone deacetylases (HDACs) have essential roles in regulating the osteogenic differentiation of bone marrow stromal cells (BMSCs) and bone formation. However, little is known regarding what regulates HDAC expression and therefore the osteogenic differentiation of BMSCs during osteogenesis. In this study, we investigated whether mechanical loading regulates HDAC expression directly and examined the role of HDACs in mechanical loading-triggered osteogenic differentiation and bone formation. We first studied the microarrays of samples from patients with osteoporosis and found that the NOTCH pathway and skeletal development gene sets were downregulated in the BMSCs of patients with osteoporosis. Then we demonstrated that mechanical stimuli can regulate osteogenesis and bone formation both in vivo and in vitro. NOTCH signaling was upregulated during cyclic mechanical stretch (CMS)-induced osteogenic differentiation, whereas HDAC1 protein expression was downregulated. The perturbation of HDAC1 expression also had a significant effect on matrix mineralization and JAG1-mediated Notch signaling, suggesting that HDAC1 acts as an endogenous attenuator of Notch signaling in the mechanotransduction of BMSCs. Chromatin immunoprecipitation (ChIP) assay results suggest that HDAC1 modulates the CMS-induced histone H3 acetylation level at the JAG1 promoter. More importantly, we found an inhibitory role of Hdac1 in regulating bone formation in response to hindlimb unloading in mice, and pretreatment with an HDAC1 inhibitor partly rescued the osteoporosis caused by mechanical unloading. Our results demonstrate, for the first time, that mechanical stimulation orchestrates genes expression involved in the osteogenic differentiation of BMSCs via the direct regulation of HDAC1, and the therapeutic inhibition of HDAC1 may be an efficient strategy for enhancing bone formation under mechanical stimulation.

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

    SciTech Connect

    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

    2014-01-24

    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.

  10. Administration of bone marrow stromal cells in sepsis attenuates sepsis-related coagulopathy.

    PubMed

    Tan, Lifei; Huang, Yueyue; Pan, Xiaojun; Quan, Shichao; Xu, Shunyao; Li, Dequan; Song, Lijun; Zhang, Xiaomin; Chen, Wanzhou; Pan, Jingye

    2016-01-01

    Coagulopathy plays an important role in sepsis. The aim of this study was to determine whether bone marrow stromal cell (BMSC) administration could attenuate coagulopathy in sepsis. In vitro: endothelial cells were cultured with/without BMSCs for 6 h following LPS stimulation and were collected for thrombomodulin (TM) and endothelial protein C receptor (EPCR) measurements. In vivo: Thirty-six mice were randomized into sham, sepsis, and sepsis + BMSC groups (n = 12 each group). Sepsis was induced through cecal ligation and puncture (CLP). BMSC infusion was started at 6 h after CLP. Lung tissues and plasma samples were collected at 24 h after CLP for enzyme-linked immunosorbent assay (ELISA), quantitative real-time RT-PCR, western blot, and immunohistochemistry analysis. In vitro: BMSCs attenuated the decrease in TM and EPCR mRNA and protein expression levels in LPS-stimulated endothelial cells. In vivo: BMSC treatment decreased lung injury and mesenteric perfusion impairment, and ameliorated coagulopathy, as suggested by the reduction in elevated TF, vWF, and TAT circulation levels. BMSC infusion decreased TF mRNA transcription and protein expression levels in lung tissues, and increased TM and EPCR mRNA transcription and expression levels. BMSC administration attenuated coagulopathy, and decreased lung injury and mesenteric perfusion impairment in sepsis. Key messages BMSCs increased the expression of TM and EPCR from endothelium cells exposed to LPS in vitro. BMSC treatment attenuated lung injury and coagulopathy in the mice cecal ligation and puncture (CLP) model. BMSC administration-attenuated coagulopathy is related to the reduced expression of TF and increased expression of TM and EPCR.

  11. Functional characterisation of bone marrow-derived mesenchymal stromal cells from COPD patients

    PubMed Central

    Roelofs, Helene; Zarcone, Maria C.; Taube, Christian; Stolk, Jan; Hiemstra, Pieter S.

    2016-01-01

    Autologous bone marrow-derived mesenchymal stromal cells (BM-MSCs) are evaluated for clinical use in chronic obstructive pulmonary disease (COPD) patients, but it is unclear whether COPD affects BM-MSCs. To investigate this, BM-MSCs from nine COPD patients and nine non-COPD age-matched controls were compared with regard to immunophenotype, growth and differentiation potential, and migration capacity. Other functional assays included the response to pro-inflammatory stimuli and inducers of the nuclear factor (erythroid derived 2)-like 2 antioxidant response element (Nrf2-ARE) pathway, and effects on NCI-H292 airway epithelial cells. No significant differences were observed in terms of morphology, proliferation and migration, except for increased adipocyte differentiation potential in the COPD group. Both groups were comparable regarding mRNA expression of growth factors and inflammatory mediators, and in their potential to induce mRNA expression of epidermal growth factor receptor ligands in NCI-H292 airway epithelial cells. MSCs from COPD patients secreted more interleukin-6 in response to pro-inflammatory stimuli. Activation of the Nrf2-ARE pathway resulted in a comparable induction of mRNA expression of four target genes, but the expression of the NAD(P)H:quinone oxidoreductase 1 gene NQO1 was lower in MSCs from COPD patients. The observation that MSCs from COPD patients are phenotypically and functionally comparable to those from non-COPD controls implies that autologous MSCs can be considered for use in the setting of clinical trials as a treatment for COPD. PMID:27730190

  12. Quantitative activation suppression assay to evaluate human bone marrow-derived mesenchymal stromal cell potency.

    PubMed

    Salem, Bahey; Miner, Samantha; Hensel, Nancy F; Battiwalla, Minoo; Keyvanfar, Keyvan; Stroncek, David F; Gee, Adrian P; Hanley, Patrick J; Bollard, Catherine M; Ito, Sawa; Barrett, A John

    2015-12-01

    With the increasing use of cell therapies involving immune modulatory cells, there is a need for a simple standardized method to evaluate and compare the suppressive potency of different cell products. We used the Karpas 299 (K299) cell line as the reference suppressor cell to develop a standardized suppression assay to quantify the immune-modulatory capacity of bone marrow-derived mesenchymal stromal cells (BM-MSCs). Healthy donor CD4 T cells were co-cultured with the K299 cell line or with third-party BM-MSCs. After stimulation with anti-CD3/CD28 beads, CD154 activation and proliferation of CD4 T cells were measured to calculate suppression. The K299 cell line reproducibly suppressed both the activation and proliferation of healthy donor CD4 T cells in a dose-dependent manner. A rapid (16-h) assay that was based on activation-suppression was selected for development. In replicate testing, there was an inherent variability of suppression of 11% coefficient of variation between different responder T cells. Suppression by BM-MSCs on different responders correlated with suppression by K299. We therefore used K299 suppression as the reference to define suppression potency of BM-MSCs in K299 Suppression Units. We found that inter-donor variability, passage number, method of manufacture and exposure of BM-MSCs to steroids or interferon-γ all affected BM-MSC potency of suppression. This method provides a platform for standardizing suppressor function to facilitate comparisons between laboratories and for use as a cell product release assay. Published by Elsevier Inc.

  13. Mechanical stimulation orchestrates the osteogenic differentiation of human bone marrow stromal cells by regulating HDAC1

    PubMed Central

    Wang, J; Wang, C D; Zhang, N; Tong, W X; Zhang, Y F; Shan, S Z; Zhang, X L; Li, Q F

    2016-01-01

    Mechanical stimulation and histone deacetylases (HDACs) have essential roles in regulating the osteogenic differentiation of bone marrow stromal cells (BMSCs) and bone formation. However, little is known regarding what regulates HDAC expression and therefore the osteogenic differentiation of BMSCs during osteogenesis. In this study, we investigated whether mechanical loading regulates HDAC expression directly and examined the role of HDACs in mechanical loading-triggered osteogenic differentiation and bone formation. We first studied the microarrays of samples from patients with osteoporosis and found that the NOTCH pathway and skeletal development gene sets were downregulated in the BMSCs of patients with osteoporosis. Then we demonstrated that mechanical stimuli can regulate osteogenesis and bone formation both in vivo and in vitro. NOTCH signaling was upregulated during cyclic mechanical stretch (CMS)-induced osteogenic differentiation, whereas HDAC1 protein expression was downregulated. The perturbation of HDAC1 expression also had a significant effect on matrix mineralization and JAG1-mediated Notch signaling, suggesting that HDAC1 acts as an endogenous attenuator of Notch signaling in the mechanotransduction of BMSCs. Chromatin immunoprecipitation (ChIP) assay results suggest that HDAC1 modulates the CMS-induced histone H3 acetylation level at the JAG1 promoter. More importantly, we found an inhibitory role of Hdac1 in regulating bone formation in response to hindlimb unloading in mice, and pretreatment with an HDAC1 inhibitor partly rescued the osteoporosis caused by mechanical unloading. Our results demonstrate, for the first time, that mechanical stimulation orchestrates genes expression involved in the osteogenic differentiation of BMSCs via the direct regulation of HDAC1, and the therapeutic inhibition of HDAC1 may be an efficient strategy for enhancing bone formation under mechanical stimulation. PMID:27171263

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

    PubMed

    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.

  15. Endogenous mesenchymal stromal cells in bone marrow are required to preserve muscle function in mdx mice.

    PubMed

    Fujita, Ryo; Tamai, Katsuto; Aikawa, Eriko; Nimura, Keisuke; Ishino, Saki; Kikuchi, Yasushi; Kaneda, Yasufumi

    2015-03-01

    The physiological role of "endogenous" bone marrow (BM) mesenchymal stromal cells (MSCs) in tissue regeneration is poorly understood. Here, we show the significant contribution of unique endogenous BM-MSC populations to muscle regeneration in Duchenne muscular dystrophy (DMD) mice (mdx). Transplantation of BM cells (BMCs) from 10-week-old mdx into 3-4-week-old mdx mice increased inflammation and fibrosis and reduced muscle function compared with mdx mice that received BMCs from 10-week-old wild-type mice, suggesting that the alteration of BMC populations in mdx mice affects the progression of muscle pathology. Two distinct MSC populations in BM, that is, hematopoietic lineage (Lin)(-) /ckit(-) /CD106(+) /CD44(+) and Lin(-) /ckit(-) /CD106(+) /CD44(-) cells, were significantly reduced in 10-week-old mdx mice in disease progression. The results of a whole-transcriptome analysis indicated that these two MSC populations have distinct gene expression profiles, indicating that the Lin(-) /ckit(-) /CD106(+) /CD44(+) and Lin(-) /ckit(-) /CD106(+) /CD44(-) MSC populations are proliferative- and dormant-state populations in BM, respectively. BM-derived Lin(-) /CD106(+) /CD44(+) MSCs abundantly migrated to damaged muscles and highly expressed tumor necrosis factor-alpha-stimulated gene/protein-6 (TSG-6), an anti-inflammatory protein, in damaged muscles. We also demonstrated that TSG-6 stimulated myoblast proliferation. The injection of Lin(-) /ckit(-) /CD106(+) /CD44(+) MSCs into the muscle of mdx mice successfully ameliorated muscle dysfunction by decreasing inflammation and enhancing muscle regeneration through TSG-6-mediated activities. Thus, we propose a novel function of the unique endogenous BM-MSC population, which countered muscle pathology progression in a DMD model.

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

    PubMed

    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.

  17. Bone marrow stromal cell transplantation preserves gammaaminobutyric acid receptor function in the injured spinal cord.

    PubMed

    Yano, Shunsuke; Kuroda, Satoshi; Shichinohe, Hideo; Seki, Toshitaka; Ohnishi, Takako; Tamagami, Hiroshi; Hida, Kazutoshi; Iwasaki, Yoshinobu

    2006-11-01

    A surprising shortage of information surrounds the mechanisms by which bone marrow stromal cells (BMSC) restore lost neurologic functions when transplanted into the damaged central nervous system. In the present study, we sought to elucidate whether BMSCs express the neuron-specific gamma-aminobutyric acid (GABA) receptor when transplanted into injured spinal cord. To examine this, we harvested and cultured rat femoral BMSCs. We then subjected Sprague-Dawley rats to thoracic spinal cord injury (SCI) with a pneumatic impact device. Fluorescence-labeled BMSCs (n = 7) were transplanted stereotactically or the vehicle in which these cells were cultured (n = 4) was introduced stereotactically into the rostral site of SCI at 7 days after injury. We evaluated GABA receptor function by measuring the binding potential for 125I-iomazenil (125I-IMZ) through in vitro autoradiography at 4 weeks after BMSC transplantation and simultaneously examined the fate of the transplanted BMSCs by immunocytochemistry. We found that the transplanted BMSC migrated toward the core of the injury and were densely distributed in the marginal region at 4 weeks after transplantation. BMSC transplantation significantly increased the binding potential for 125I-IMZ (p = 0.0376) and increased the number of GABA receptor-positive cells (p = 0.0077) in the marginal region of the injury site. Some of the transplanted BMSCs were positive for microtubule-associated protein-2 and the alpha1 subunit of GABA(A) receptor in the region of injury. These findings suggest that BMSCs have the potential to support the survival of neurons in the marginal region of SCI and can partly differentiate into neurons, regenerating spinal cord tissue at the site of injury.

  18. Bone Marrow-Derived Mesenchymal Stromal Cells from Patients with Sickle Cell Disease Display Intact Functionality.

    PubMed

    Stenger, Elizabeth O; Chinnadurai, Raghavan; Yuan, Shala; Garcia, Marco; Arafat, Dalia; Gibson, Greg; Krishnamurti, Lakshmanan; Galipeau, Jacques

    2017-01-26

    Hematopoietic cell transplantation (HCT) is the only cure for sickle cell disease (SCD), but engraftment remains challenging in patients lacking matched donors. Infusion of mesenchymal stromal cells (MSCs) at the time of HCT may promote hematopoiesis and ameliorate graft-versus-host disease. Experimental murine models suggest MSC major histocompatibility complex compatibility with recipient impacts their in vivo function, suggesting autologous MSCs could be superior to third-party MSCs for promoting HCT engraftment. Here we tested whether bone marrow (BM)-derived MSCs from SCD subjects have comparable functionality compared with MSCs from healthy volunteers. SCD MSC doubling time and surface marker phenotype did not differ significantly from non-SCD. Third-party and autologous (SCD) T cell proliferation was suppressed in a dose-dependent manner by all MSCs. SCD MSCs comparably expressed indoleamine-2,3-dioxygenase, which based on transwell and blocking experiments appeared to be the dominant immunomodulatory pathway. The expression of key genes involved in hematopoietic stem cell (HSC)-MSC interactions was minimally altered between SCD and non-SCD MSCs. Expression was, however, altered by IFN-γ stimulation, particularly CXCL14, CXCL26, CX3CL1, CKITL, and JAG1, indicating the potential to augment MSC expression by cytokine stimulation. These data demonstrate the feasibility of expanding BM-derived MSCs from SCD patients that phenotypically and functionally do not differ per International Society of Cell Therapy essential criteria from non-SCD MSCs, supporting initial evaluation (primarily for safety) of autologous MSCs to enhance haploidentical HSC engraftment in SCD.

  19. Prostaglandin E2 impairs osteogenic and facilitates adipogenic differentiation of human bone marrow stromal cells.

    PubMed

    Noack, Carolin; Hempel, Ute; Preissler, Carolin; Dieter, Peter

    2015-03-01

    The synthetic glucocorticoid dexamethasone (dex) is a mandatory additive to induce osteogenic differentiation of bone marrow stromal cell (BMSC) in vitro; however it is also known to promote the pathogenesis of osteoporotic bone disease in vivo. In this study human (h)BMSC were cultured in osteogenic medium containing β-glycerophosphate and ascorbate (OM) and in OM containing dex (OM/D). It was seen that dex induced in human (h)BMSC both, osteogenic and adipogenic differentiation markers. Dex reveals its anti-inflammatory effect by reducing endogenous prostaglandin E2 (PGE2) formation and by suppressing the inducible enzymes cyclooxygenase 2 and microsomal PGE2 synthase 1. It was further seen that dex enhanced the expression of prostaglandin receptors, mainly EP2 and EP4 receptor subtypes. We thus hypothesized that dex enforces the susceptibility of hBMSC to respond to exogenous PGE2. Permanent exposure of hBMSC which were cultured in OM/D to PGE2, decreased osteogenic and increased adipogenic differentiation markers. The effects of PGE2 were preferentially mediated by receptor subtypes EP2 and EP4; EP1 was partially involved in pro-adipogenic effects, and EP3 was partially involved in anti-osteogenic effects. These results suggest that dex suppresses the formation of endogenous PGE2 but also enables hBMSC to respond to PGE2 due to the induction of PGE2 receptors EP2 and EP4. PGE2 then shifts in hBMSC the balance from osteogenic to adipogenic differentiation.

  20. Heparin affects human bone marrow stromal cell fate: Promoting osteogenic and reducing adipogenic differentiation and conversion.

    PubMed

    Simann, Meike; Schneider, Verena; Le Blanc, Solange; Dotterweich, Julia; Zehe, Viola; Krug, Melanie; Jakob, Franz; Schilling, Tatjana; Schütze, Norbert

    2015-09-01

    Heparins are broadly used for the prevention and treatment of thrombosis and embolism. Yet, osteoporosis is considered to be a severe side effect in up to one third of all patients on long-term treatment. However, the mechanisms underlying this clinical problem are only partially understood. To investigate if heparin affects differentiation of skeletal precursors, we examined the effects of heparin on the osteogenic and adipogenic lineage commitment and differentiation of primary human bone marrow stromal cells (hBMSCs). Due to the known inverse relationship between adipogenesis and osteogenesis and the capacity of pre-differentiated cells to convert into the respective other lineage, we also determined heparin effects on osteogenic conversion and adipogenic differentiation/conversion. Interestingly, heparin did not only significantly increase mRNA expression and enzyme activity of the osteogenic marker alkaline phosphatase (ALP), but it also promoted mineralization during osteogenic differentiation and conversion. Furthermore, the mRNA expression of the osteogenic marker bone morphogenic protein 4 (BMP4) was enhanced. In addition, heparin administration partly prevented adipogenic differentiation and conversion demonstrated by reduced lipid droplet formation along with a decreased expression of adipogenic markers. Moreover, luciferase reporter assays, inhibitor experiments and gene expression analyses revealed that heparin had putative permissive effects on osteogenic signaling via the BMP pathway and reduced the mRNA expression of the Wnt pathway inhibitors dickkopf 1 (DKK1) and sclerostin (SOST). Taken together, our data show a rather supportive than inhibitory effect of heparin on osteogenic hBMSC differentiation and conversion in vitro. Further studies will have to investigate the net effects of heparin administration on bone formation versus bone resorption in vivo to unravel the molecular mechanisms of heparin-associated osteoporosis and reconcile

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

    PubMed Central

    Hassan, Nathaniel; Mengatto, Cristiane M.; Langfelder, Peter; Hokugo, Akishige; Tahara, Yu; Colwell, Christopher S.

    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. PMID:28817668

  2. Human marrow stromal cell therapy for stroke in rat: neurotrophins and functional recovery.

    PubMed

    Li, Y; Chen, J; Chen, X G; Wang, L; Gautam, S C; Xu, Y X; Katakowski, M; Zhang, L J; Lu, M; Janakiraman, N; Chopp, M

    2002-08-27

    To test the effect of i.v.-injected human bone marrow stromal cells (hMSC) on neurologic functional deficits after stroke in rats. Rats were subjected to transient middle cerebral artery occlusion and IV injected with 3 x 10(6) hMSC 1 day after stroke. Functional outcome was measured before and 1, 7, and 14 days after stroke. Mixed lymphocyte reaction and the development of cytotoxic T lymphocytes measured the immune rejection of hMSC. A monoclonal antibody specific to human cellular nuclei (mAb1281) was used to identify hMSC and to measure neural phenotype. ELISA analyzed neurotrophin levels in cerebral tissue from hMSC-treated or nontreated rats. Bromodeoxyuridine injections were used to identify newly formed cells. Significant recovery of function was found in rats treated with hMSC at 14 days compared with control rats with ischemia. Few (1 to 5%) hMSC expressed proteins phenotypic of brain parenchymal cells. Brain-derived neurotrophic factor and nerve growth factor significantly increased, and apoptotic cells significantly decreased in the ischemic boundary zone; significantly more bromodeoxyuridine-reactive cells were detected in the subventricular zone of the ischemic hemisphere of rats treated with hMSC. hMSC induced proliferation of lymphocytes without the induction of cytotoxic T lymphocytes. Neurologic benefit resulting from hMSC treatment of stroke in rats may derive from the increase of growth factors in the ischemic tissue, the reduction of apoptosis in the penumbral zone of the lesion, and the proliferation of endogenous cells in the subventricular zone.

  3. Mesenchymal stromal cells derived from CD271(+) bone marrow mononuclear cells exert potent allosuppressive properties.

    PubMed

    Kuçi, Zyrafete; Kuçi, Selim; Zircher, Susanne; Koller, Stefanie; Schubert, Ralf; Bönig, Halvard; Henschler, Reinhard; Lieberz, Ralf; Klingebiel, Thomas; Bader, Peter

    2011-11-01

    BACKGROUND AIMS. Because data on the immunosuppressive effect of different subsets of mesenchymal stromal cells (MSC) are sparse, we investigated the molecular and cellular mechanisms underlying the allosuppressive effect of MSC generated from bone marrow CD271(+) cells (CD271-MSC) and asked whether this potential is comparable with that of MSC generated through plastic adherence (PA-MSC). METHODS. The immunosuppressive effect of CD271-MSC on the allogeneic reaction was investigated by mixed lymphocyte reaction (MLR). RESULTS. CD271-MSC significantly suppressed the alloantigen-induced proliferation of mononuclear cells (MNC) of two HLA-disparate donors at all MSC:MNC ratios, 1:1, 1:2 and 1:10. They also demonstrated a significantly higher allosuppression than PA-MSC at an MSC:MNC ratio of 1:1. This inhibitory effect was associated with significantly elevated levels of prostaglandin E2 (PGE2) at ratios of 1:1 and 1:2 (about 4-fold), but not at a ratio of 1:10. Indomethacin, and inhibitor of cyclooxygenase-1 and 2 necessary for the biosynthesis of PGE2, mitigated suppressive effects of CD271-MSC only at a ratio of 1:1, indicating that PGE2 is not involved in MSC-mediated inhibition when allogeneic MNC are in excess. The increase of PGE2 was associated with a significant decrease of pro-inflammatory cytokine levels (interferon-gamma and tumor necrosis-alpha), while no changes in levels of interleukin-10, soluble HLA-G and nitric oxide were observed. In addition, CD271-MSC induced an expansion of highly suppressive naive CD4(+)CD25(high)CD45RA(+)CD62L(+) T-regulatory cells, which may extend their allosuppressive effect. CONCLUSIONS. Our data suggest that CD271-MSC exert potent allosuppressive properties and therefore can be used as a reasonable alternative to PA-MSC for the treatment of patients with graft-versus-host disease.

  4. Molecular cloning and chromosomal mapping of bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-cell growth

    SciTech Connect

    Ishikawa, Jun; Kaisho, Tsuneyasu; Tomizawa, Hitoshi

    1995-04-10

    Bone marrow stromal cells regulate B-cell growth and development through their surface molecules and cytokines. In this study, we generated a mAb, RS38, that recognized a novel human membrane protein, BST-2, expressed on bone marrow stromal cell lines and synovial cell lines. We cloned a cDNA encoding BST-2 from a rheumatoid arthritis-derived synovial cell line. BST-2 is a 30- to 36-kDa type II transmembrane protein, consisting of 180 amino acids. The BST-2 gene (HGMW-approved symbol BST2) is located on chromosome 19p13.2. BST-2 is expressed not only on certain bone marrow stromal cell lines but also on various normal tissues, although its expression pattern is different from that of another bone marrow stromal cell surface molecule, BST-1. BST-2 surface expression on fibroblast cell lines facilitated the stromal cell-dependent growth of a murine bone marrow-derived pre-B-cell line, DW34. The results suggest that BST-2 may be involved in pre-B-cell growth. 45 refs., 7 figs., 2 tabs.

  5. Quantitative analysis of glycans, related genes, and proteins in two human bone marrow stromal cell lines using an integrated strategy.

    PubMed

    Li, Xiang; Li, Dongliang; Pang, Xingchen; Yang, Ganglong; Deeg, H Joachim; Guan, Feng

    2015-09-01

    Altered expression of glycans is associated with cell-cell signal transduction and regulation of cell functions in the bone marrow micro-environment. Studies of this micro-environment often use two human bone marrow stromal cell lines, HS5 and HS27a, co-cultured with myeloid cells. We hypothesized that differential protein glycosylation between these two cell lines may contribute to functional differences in in vitro co-culture models. In this study, we applied an integrated strategy using genomic, proteomic, and functional glycomic techniques for global expression profiling of N-glycans and their related genes and enzymes in HS5 cells versus HS27a cells. HS5 cells had significantly enhanced levels of bisecting N-glycans (catalyzed by MGAT3 [β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase]), whereas HS27a cells had enhanced levels of Galβ1,4GlcNAc (catalyzed by β4GalT1 [β4-galactosyltransferase I]). This integrated strategy provides useful information regarding the functional roles of glycans and their related glycogenes and glycosyltransferases in the bone marrow microenvironment, and a basis for future studies of crosstalk among stromal cells and myeloma cells in co-culture.

  6. Human Bone Marrow Stromal Cells Differentiate Into Corneal Tissue and Prevent Ocular Graft-Versus-Host Disease in Mice.

    PubMed

    Sánchez-Abarca, Luis Ignacio; Hernández-Galilea, Emiliano; Lorenzo, Rebeca; Herrero, Carmen; Velasco, Almudena; Carrancio, Soraya; Caballero-Velázquez, Teresa; Rodríguez-Barbosa, José Ignacio; Parrilla, Marta; Del Cañizo, Consuelo; San Miguel, Jesús; Aijón, José; Pérez-Simón, José Antonio

    2015-01-01

    Clinical trials have assessed the use of human bone marrow stromal cells (hBMSCs) for the treatment of immune-related disorders such as graft-versus-host disease (GVHD). In the current study, we show that GFP(+)-transduced hBMSCs generated from bone marrow migrate and differentiate into corneal tissue after subconjunctival injection in mice. Interestingly, these hBMSCs display morphological features of epithelial, stromal, and endothelial cells and appear at different layers and with different morphologies depending on their position within the epithelium. Furthermore, these cells display ultrastructural properties, such as bundles of intermediate filaments, interdigitations, and desmosomes with GFP(-) cells, which confirms their differentiation into corneal tissues. GFP(+)-transduced hBMSCs were injected at different time points into the right eye of lethally irradiated mice undergoing bone marrow transplantation, which developed ocular GVHD (oGVHD). Remarkably, hBMSCs massively migrate to corneal tissues after subconjunctival injection. Both macroscopic and histopathological examination showed minimal or no evidence of GVHD in the right eye, while the left eye, where no hBMSCs were injected, displayed features of GVHD. Thus, in the current study, we confirm that hBMSCs may induce their therapeutic effect at least in part by differentiation and regeneration of damaged tissues in the host. Our results provide experimental evidence that hBMSCs represent a potential cellular therapy to attenuate oGVHD.

  7. Autologous bone marrow stromal cells are promising candidates for cell therapy approaches to treat bone degeneration in sickle cell disease.

    PubMed

    Lebouvier, Angélique; Poignard, Alexandre; Coquelin-Salsac, Laura; Léotot, Julie; Homma, Yasuhiro; Jullien, Nicolas; Bierling, Philippe; Galactéros, Frédéric; Hernigou, Philippe; Chevallier, Nathalie; Rouard, Hélène

    2015-11-01

    Osteonecrosis of the femoral head is a frequent complication in adult patients with sickle cell disease (SCD). To delay hip arthroplasty, core decompression combined with concentrated total bone marrow (BM) treatment is currently performed in the early stages of the osteonecrosis. Cell therapy efficacy depends on the quantity of implanted BM stromal cells. For this reason, expanded bone marrow stromal cells (BMSCs, also known as bone marrow derived mesenchymal stem cells) can be used to improve osteonecrosis treatment in SCD patients. In this study, we quantitatively and qualitatively evaluated the function of BMSCs isolated from a large number of SCD patients with osteonecrosis (SCD-ON) compared with control groups (patients with osteonecrosis not related to SCD (ON) and normal donors (N)). BM total nuclear cells and colony-forming efficiency values (CFE) were significantly higher in SCD-ON patients than in age and sex-matched controls. The BMSCs from SCD-ON patients were similar to BMSCs from the control groups in terms of their phenotypic and functional properties. SCD-ON patients have a higher frequency of BMSCs that retain their bone regeneration potential. Our findings suggest that BMSCs isolated from SCD-ON patients can be used clinically in cell therapy approaches. This work provides important preclinical data that is necessary for the clinical application of expanded BMSCs in advanced therapies and medical products.

  8. Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow.

    PubMed

    Zhou, Bo O; Yue, Rui; Murphy, Malea M; Peyer, James G; Morrison, Sean J

    2014-08-07

    Studies of the identity and physiological function of mesenchymal stromal cells (MSCs) have been hampered by a lack of markers that permit both prospective identification and fate mapping in vivo. We found that Leptin Receptor (LepR) is a marker that highly enriches bone marrow MSCs. Approximately 0.3% of bone marrow cells were LepR(+), 10% of which were CFU-Fs, accounting for 94% of bone marrow CFU-Fs. LepR(+) cells formed bone, cartilage, and adipocytes in culture and upon transplantation in vivo. LepR(+) cells were Scf-GFP(+), Cxcl12-DsRed(high), and Nestin-GFP(low), markers which also highly enriched CFU-Fs, but negative for Nestin-CreER and NG2-CreER, markers which were unlikely to be found in CFU-Fs. Fate-mapping showed that LepR(+) cells arose postnatally and gave rise to most bone and adipocytes formed in adult bone marrow, including bone regenerated after irradiation or fracture. LepR(+) cells were quiescent, but they proliferated after injury. Therefore, LepR(+) cells are the major source of bone and adipocytes in adult bone marrow.

  9. Leptin Receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow

    PubMed Central

    Zhou, Bo O.; Yue, Rui; Murphy, Malea M.; Peyer, James; Morrison, Sean J.

    2014-01-01

    SUMMARY Studies of the identity and physiological function of mesenchymal stromal cells (MSCs) have been hampered by a lack of markers that permit both prospective identification and fate mapping in vivo. We found Leptin Receptor (LepR) is a marker that highly enriches bone marrow MSCs. Approximately 0.3% of bone marrow cells were LepR+, 10% of which were CFU-F, accounting for 94% of bone marrow CFU-F. LepR+ cells formed bone, cartilage, and adipocytes in culture and upon transplantation in vivo. LepR+ cells were Scf-GFP+, Cxcl12-DsRedhigh, and Nestin-GFPlow, markers which also highly enriched CFU-F, but negative for Nestin-CreER and NG2-CreER, markers which included few CFU-F. Fate-mapping showed LepR+ cells arose postnatally and gave rise to most bone and adipocytes formed in adult bone marrow, including bone regenerated after irradiation or fracture. LepR+ cells were quiescent but proliferated after injury. LepR+ cells are the major source of bone and adipocytes in adult bone marrow. PMID:24953181

  10. Cyclic Tensile Culture Promotes Fibroblastic Differentiation of Marrow Stromal Cells Encapsulated in Poly(Ethylene Glycol)-Based Hydrogels

    PubMed Central

    Doroski, Derek M.; Levenston, Marc E.

    2010-01-01

    To inform future efforts in tendon/ligament tissue engineering, our laboratory has developed a well-controlled model system with the ability to alter both external tensile loading parameters and local biochemical cues to better understand marrow stromal cell differentiation in response to both stimuli concurrently. In particular, the synthetic, poly(ethylene glycol)-based hydrogel material oligo(poly(ethylene glycol) fumarate) (OPF) has been explored as a cell carrier for this system. This biomaterial can be tailored to present covalently incorporated bioactive moieties and can be loaded in our custom cyclic tensile bioreactor for up to 28 days with no loss of material integrity. Human marrow stromal cells encapsulated in these OPF hydrogels were cultured (21 days) under cyclic tensile strain (10%, 1 Hz, 3 h of strain followed by 3 h without) or at 0% strain. No difference was observed in cell number due to mechanical stimulation or across time (n = 4), with cells remaining viable (n = 4) through 21 days. Cyclic strain significantly upregulated all tendon/ligament fibroblastic genes examined (collagen I, collagen III, and tenascin-C) by day 21 (n ≥ 6), whereas genes for other pathways (osteogenic, chondrogenic, and adipogenic) did not increase. After 21 days, the presence of collagen I and tenascin-C was observed via immunostaining (n = 2). This study demonstrates the utility of this hydrogel/bioreactor system as a versatile, yet well-controlled, model environment to study marrow stromal cell differentiation toward the tendon/ligament phenotype under a variety of conditions. PMID:20666585

  11. Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells.

    PubMed

    Mathews, Smitha; Bhonde, Ramesh; Gupta, Pawan Kumar; Totey, Satish

    2012-09-01

    The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21 day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications.

  12. The HIV proteins Tat and Nef promote human bone marrow mesenchymal stem cell senescence and alter osteoblastic differentiation.

    PubMed

    Beaupere, Carine; Garcia, Marie; Larghero, Jerome; Fève, Bruno; Capeau, Jacqueline; Lagathu, Claire

    2015-08-01

    To maintain bone mass turnover and bone mineral density (BMD), bone marrow (BM) mesenchymal stem cells (MSCs) are constantly recruited and subsequently differentiated into osteoblasts. HIV-infected patients present lower BMD than non-HIV infected individuals and a higher prevalence of osteopenia/osteoporosis. In antiretroviral treatment (ART)-naive patients, encoded HIV proteins represent pathogenic candidates. They are released by infected cells within BM and can impact on neighbouring cells. In this study, we tested whether HIV proteins Tat and/or Nef could induce senescence of human BM-MSCs and reduce their capacity to differentiate into osteoblasts. When compared to nontreated cells, MSCs chronically treated with Tat and/or Nef up to 30 days reduced their proliferative activity and underwent early senescence, associated with increased oxidative stress and mitochondrial dysfunction. The antioxidant molecule N-acetyl- cysteine had no or minimal effects on Tat- or Nef-induced senescence. Tat but not Nef induced an early increase in NF-κB activity and cytokine/chemokine secretion. Tat-induced effects were prevented by the NF-κB inhibitor parthenolide, indicating that Tat triggered senescence via NF-κB activation leading to oxidative stress. Otherwise, Nef- but not Tat-treated cells displayed early inhibition of autophagy. Rapamycin, an autophagy inducer, reversed Nef-induced senescence and oxidative stress. Moreover, Tat+Nef had cumulative effects. Finally, Tat and/or Nef decreased the MSC potential of osteoblastic differentiation. In conclusion, our in vitro data show that Tat and Nef could reduce the number of available precursors by inducing MSC senescence, through either enhanced inflammation or reduced autophagy. These results offer new insights into the pathophysiological mechanisms of decreased BMD in HIV-infected patients.

  13. The HIV proteins Tat and Nef promote human bone marrow mesenchymal stem cell senescence and alter osteoblastic differentiation

    PubMed Central

    Beaupere, Carine; Garcia, Marie; Larghero, Jerome; Fève, Bruno; Capeau, Jacqueline; Lagathu, Claire

    2015-01-01

    To maintain bone mass turnover and bone mineral density (BMD), bone marrow (BM) mesenchymal stem cells (MSCs) are constantly recruited and subsequently differentiated into osteoblasts. HIV-infected patients present lower BMD than non-HIV infected individuals and a higher prevalence of osteopenia/osteoporosis. In antiretroviral treatment (ART)-naive patients, encoded HIV proteins represent pathogenic candidates. They are released by infected cells within BM and can impact on neighbouring cells. In this study, we tested whether HIV proteins Tat and/or Nef could induce senescence of human BM-MSCs and reduce their capacity to differentiate into osteoblasts. When compared to nontreated cells, MSCs chronically treated with Tat and/or Nef up to 30 days reduced their proliferative activity and underwent early senescence, associated with increased oxidative stress and mitochondrial dysfunction. The antioxidant molecule N-acetyl- cysteine had no or minimal effects on Tat- or Nef-induced senescence. Tat but not Nef induced an early increase in NF-κB activity and cytokine/chemokine secretion. Tat-induced effects were prevented by the NF-κB inhibitor parthenolide, indicating that Tat triggered senescence via NF-κB activation leading to oxidative stress. Otherwise, Nef- but not Tat-treated cells displayed early inhibition of autophagy. Rapamycin, an autophagy inducer, reversed Nef-induced senescence and oxidative stress. Moreover, Tat+Nef had cumulative effects. Finally, Tat and/or Nef decreased the MSC potential of osteoblastic differentiation. In conclusion, our in vitro data show that Tat and Nef could reduce the number of available precursors by inducing MSC senescence, through either enhanced inflammation or reduced autophagy. These results offer new insights into the pathophysiological mechanisms of decreased BMD in HIV-infected patients. PMID:25847297

  14. Deflazacort increases osteoclast formation in mouse bone marrow culture and the ratio of RANKL/OPG mRNA expression in marrow stromal cells.

    PubMed Central

    Chung, H.; Kang, Y. S.; Hwang, C. S.; Moon, I. K.; Yim, C. H.; Choi, K. H.; Han, K. O.; Jang, H. C.; Yoon, H. K.; Han, I. K.

    2001-01-01

    Information on precise effects of deflazacort on bone cell function, especially osteoclasts, is quite limited. Therefore, the present study was undertaken to test effects of deflazacort on osteoclast-like cell formation in mouse bone marrow cultures and on the regulation of osteoprotegerin (OPG) and its ligand (RANKL) mRNA expressions by RT-PCR in the ST2 marrow stromal cells. TRAP-positive mononuclear cells increased after the treatment of deflazacort at 10(-9) to 10(-7) M alone for 6 days in a dose-dependent manner. Number of TRAP-positive multi-nucleated cells (MNCs) increased significantly with combined treatment of deflazacort at 10(-7) M and 1,25-(OH)2D3 at 10(-9) M compared to that of cultures treated with 1,25-(OH)2D3 alone (p<0.05). Exposure to deflazacort at 10(-7) M in the presence of 1,25-(OH)2D3 at 10(-9) M in the last 3-day culture had greater stimulatory effect on osteoclast-like cell formation than that of the first 3-day culture did. Deflazacort at 10(-10) -10(-6) M downregulated OPG and upregulated RANKL in mRNA levels in a dose-dependent manner. These observations suggest that deflazacort stimulate osteoclast precursor in the absence of 1,25-(OH)2D3 and enhance differentiation of osteoclasts in the presence of 1,25-(OH)2D3. These effects are, in part, thought to be mediated by the regulation of the expression of OPG and RANKL mRNA in marrow stromal cells. PMID:11748360

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

    PubMed

    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.

  16. The safety of autologous and metabolically fit bone marrow mesenchymal stromal cells in medically refractory Crohn's disease - a phase 1 trial with three doses.

    PubMed

    Dhere, T; Copland, I; Garcia, M; Chiang, K Y; Chinnadurai, R; Prasad, M; Galipeau, J; Kugathasan, S

    2016-09-01

    Mesenchymal stromal cells ability to reset immune functionalities may be useful in Crohn's disease. To perform a first-in-human phase 1 safety clinical trial of metabolically fit autologous bone marrow-derived mesenchymal stromal cells in 12 subjects with Crohn's disease utilising three doses. Autologous mesenchymal stromal cells were derived from marrow aspirate and propagated for 2-3 weeks with fibrinogen depleted human platelet lysate and subsequently administered to subjects without interval cryobanking. Twelve subjects received a single mesenchymal stromal cell intravenous infusion of 2, 5 or 10 million cells/kg BW(n = 4/group). Infused mesenchymal stromal cells were analysed for cell surface marker expression, IDO(indoleamine 2,3-dioxygenase) upregulation by IFNγ stimulation, and inhibition of third party peripheral blood mononuclear cell proliferation in vitro. The primary end point measured was safety and tolerability; clinical response was assessed as a secondary endpoint. All patients tolerated the mesenchymal stromal cell infusion well and no dose limiting toxicity was seen. Seven patients had serious adverse events of which five were hospitalisations for Crohn's disease flare. Two of these serious adverse events were possibly related to the mesenchymal stromal cells infusion. Five subjects showed clinical response 2 weeks after the infusion. Mesenchymal stromal cell phenotype, cytokine responsiveness, and peripheral blood mononuclear cell proliferation blockade were not different among the patients. Single infusion of fresh autologous bone marrow mesenchymal stromal cells propagated ex vivo using human platelet lysate-supplemented media was safe and feasible at intravenous doses of up to 10 million cells/kg BW in patients with Crohn's disease. © 2016 John Wiley & Sons Ltd.

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

    PubMed

    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

  18. Material Properties and Osteogenic Differentiation of Marrow Stromal Cells on Fiber-Reinforced Laminated Hydrogel Nanocomposites

    PubMed Central

    Xu, Weijie; Ma, Junyu; Jabbari, Esmaiel

    2009-01-01

    The fibrils in the bone matrix are glued together by ECM proteins to form laminated structures (osteons) to provide elasticity and a supportive substrate for osteogenesis. The objective of this work was to investigate material properties and osteogenic differentiation of bone marrow stromal (BMS) cells seeded on osteon-mimetic fiber-reinforced hydrogel/apatite composites. Layers of electrospun poly(L-lactide) (L-PLA) fiber mesh coated with a poly(lactide-co-ethylene oxide fumarate) (PLEOF) hydrogel precursor solution were stacked and pressed together, and crosslinked to produce a laminated fiber-reinforced composite. Hydroxyapatite (HA) nanocrystals were added to the precursor solution to produce an osteoconductive matrix for BMS cells. Acrylamide-terminated RGD peptide (Ac-GRGD) was conjugated to the PLEOF/HA hydrogel phase to promote focal point adhesion of BMS cells. Laminates were characterized with respect to Young’s modulus, degradation kinetics, and osteogenic differentiation of BMS cells. The moduli of the laminates under dry and wet conditions were significantly higher than those of the fiber mesh and PLEOF/HA hydrogel, and within the range of values reported for wet human cancellous bone. At days 14 and 21, ALPase activity of the laminates was significantly higher than those of the fiber mesh and hydrogel. Lamination significantly increased the extent of mineralization of BMS cells and laminates with HA and conjugated with RGD (Lam-RGD-HA) had 2.7-, 3.5-, and 2.8-fold higher calcium content (compared to laminates without HA or RGD) after 7, 14, and 21 days, respectively. The Lam-RGD-HA group had significantly higher expression of osteopontin (OP) and osteocalcin (OC) compared to the hydrogel or laminates without HA or RGD, consistent with the higher ALPase activity and calcium content of Lam-RGD-HA. Laminated osteon-mimetic structures have the potential to provide mechanical strength to the regenerating region as well as supporting the differentiation

  19. Repopulation of Intrasynovial Flexor Tendon Allograft with Bone Marrow Stromal Cells: An Ex Vivo Model

    PubMed Central

    Amadio, Peter C.; Thoreson, Andrew R.; An, Kai-Nan

    2014-01-01

    Purpose: Delayed healing is a common problem whenever tendon allografts are used for tendon or ligament reconstruction. Repopulating the allograft with host cells may accelerate tendon regeneration, but cell penetration into the allograft tendon is limited. Processing the tendon surface with slits that guide cells into the allograft substrate may improve healing. The purpose of this study was to describe a surface modification of allograft tendon that includes slits to aid cell repopulation and lubrication to enhance tendon gliding. Methods: Canine flexor digitorum profundus tendons were used for this study. Cyclic gliding resistance was measured over 1000 cycles. Tensile stiffness was assessed for normal tendon, tendon decellularized with trypsin and Triton X-100 (decellularized group), tendon decellularized and perforated with multiple slits (MS group) and tendon decellularized, perforated with slits and treated with a carbodiimide-derivatized hyaluronic acid and gelatin (cd-HA-gelatin) surface modification (MS-SM group). To assess tendon repopulation, bone marrow stromal cells (BMSCs) were used in the decellularized and MS groups. DNA concentration and histology were evaluated and compared to normal tendons and nonseeded decellularized tendons. Results: The gliding resistance of the decellularized and MS groups was significantly higher compared with the normal group. There was no significant difference in gliding resistance between the decellularized and MS group. Gliding resistance of the normal group and MS-SM group was not significantly different. The Young's modulus was not significantly different among the four groups. The DNA concentration in the MS group was significantly lower than in normal tendons, but significantly higher than in decellularized tendons, with or without BMSCs. Viable BMSCs were found in the slits after 2 weeks in tissue culture. Conclusions: Tendon slits can successfully harbor BMSCs without compromising their survival and without

  20. Repair of goat tibial defects with bone marrow stromal cells and beta-tricalcium phosphate.

    PubMed

    Liu, Guangpeng; Zhao, Li; Zhang, Wenjie; Cui, Lei; Liu, Wei; Cao, Yilin

    2008-06-01

    Tissue engineering techniques have been proven effective in bone regeneration and repairing load-bearing bone defects. Previous studies, however, have heretofore been limited to the use of slowdegradable or natural biomaterials as scaffolds. There are, however, no reports on using biodegradable, synthetic beta-tricalcium phosphate (beta-TCP) as scaffolds to repair weight-bearing bone defects in large animals. In the present study, highly porous beta-TCP scaffolds prepared by the polymeric sponge method were used to repair goat tibial defects. Fifteen goats were randomly assigned to one of three groups, and a 26 mm-long defect at the middle part of the right tibia in each goat was created. In Group A (six goats), a porous beta-TCP ceramic cylinder that had been loaded with osteogenically induced autologous bone marrow stromal cells (BMSCs) was implanted in the defect of each animal. In Group B (six goats), the same beta-TCP ceramic cylinder without any cells loaded was placed in the defect. In Group C (three goats), the defect was left untreated. In Group A, bony union can be observed by gross view, X-ray and micro-computed tomography (Micro-CT) detection, and histological observation at 32 weeks post-implantation. The implanted beta-TCP scaffolds were almost completely replaced by tissue-engineered bone. Bone mineral density in the repaired area of Group A was significantly higher (p < 0.05) than that of Group B, in which scant new bone was formed in each defect and the beta-TCP hadn't been completely resorbed at 32 weeks. Moreover, the tissue-engineered bone of Group A had similar biomechanical properties as that of the normal left tibia in terms of bending strength and Young's modulus (p > 0.05). In Group C, little or no new bone was formed, and non-union occurred, showing that the 26 mm segmental defect of the goat tibia was critical sized at 32 weeks. Thus, it can be concluded that the mechanical properties of the BMSCs/beta-TCP composites could be much

  1. A novel synthetic peptide vector system for optimal gene delivery to bone marrow stromal cells.

    PubMed

    Haitao, Pan; Qixin, Zheng; Xiaodong, Guo

    2007-03-01

    A 23-amino acid, bifunctional, integrin-targeted synthetic peptide was evaluated for ex vivo gene delivery to rabbit bone marrow stromal cells (BMSCs). The peptide (K)(16)GRGDSPC consists of an amino terminal domain of 16 lysines for electrostatic binding of DNA, and a 7-amino acid integrin-binding domain at the carboxyl terminal. PcDNA3-EGFP plasmids were transfected into BMSCs by (K)(16)GRGDSPC and the positive cells gave out a bright green fluorescence. High levels of gene delivery of pcDNA3-TGF-beta1 plasmids were obtained with 2 to 4 microg/ml DNA concentration, with (K)(16)GRGDSPC at an optimal peptide: DNA w/w ratio of 3:1, with a required exposure time of more than 4 h but shorter than 24 h for BMSC exposure to the peptide/DNA complexes with completely absent serum in the initial stage; with 100 microM chloroquine and at least 8 h exposure for BMSC exposure to chloroquine; with a fusogenic peptide at an optimal (K)(16)GRGDSPC/DNA/fusogenic peptide w/w ratio of 3:1:5; and with Lipofectamine 2000 at an optimal (K)(16)GRGDSPC/DNA/Lipofectamine 2000 w/w ratio of 3:1:2 at a constant DNA concentration of 2 microg/ml. Chloroquine, the fusogenic peptide and Lipofectamine 2000 all significantly promoted gene delivery, but chloroquine was more effective than the fusogenic peptide and had obvious synergistic effects with Lipofectamine 2000. Under optimal conditions, TGF-beta1 gene was transfected into BMSCs without observable toxicity, and the stable expression was examined by RT-PCR and Western blot analysis. The stable transgenic cells showed obvious bands. This novel synthetic peptide, providing a new way for the use of polylysine and RGD motif in DNA vector system, is potentially well suited to ex vivo gene delivery to BMSCs for experimental and clinical applications in the field of bone tissue engineering.

  2. Modulation properties of factors released by bone marrow stromal cells on activated microglia: an in vitro study

    PubMed Central

    Cizkova, Dasa; Devaux, Stéphanie; Le Marrec-Croq, Françoise; Franck, Julien; Slovinska, Lucia; Blasko, Juraj; Rosocha, Jan; Spakova, Timea; Lefebvre, Christophe; Fournier, Isabelle; Salzet, Michel

    2014-01-01

    In the present paper we develop a new non-cell based (cell-free) therapeutic approach applied to BV2 microglial cells and spinal cord derived primary microglia (PM) using conditioned media from rat bone marrow stromal cells (BMSCs-CM). First we collected conditioned media (CM) from either naive or injured rat spinal cord tissue (SCI-CM, inflammatory stimulation agent) and from rat bone marrow stromal cells (BMSCs-CM, therapeutic immunomodulation agent). They were both subsequently checked for the presence of chemokines and growth, neurotrophic and neural migration factors using proteomics analysis. The data clearly showed that rat BMSCs-CM contain in vitro growth factors, neural migration factors, osteogenic factors, differentiating factors and immunomodulators, whereas SCI-CM contain chemokines, chemoattractant factors and neurotrophic factors. Afterwards we determined whether the BMSCs-CM affect chemotactic activity, NO production, morphological and pro-apoptotic changes of either BV2 or PM cells once activated with SCI-CM. Our results confirm the anti-migratory and NO-inhibitory effects of BMSCs-CM on SCI-CM-activated microglia with higher impact on primary microglia. The cytotoxic effect of BMSCs-CM occurred only on SCI-CM-stimulated BV2 cells and PM, not on naive BV2 cells, nor on PM. Taken together, the molecular cocktail found in BMSCs-CM is favorable for immunomodulatory properties. PMID:25524416

  3. Formation and differentiation of three-dimensional rat marrow stromal cell culture on microcarriers in a rotating-wall vessel

    NASA Technical Reports Server (NTRS)

    Qiu, Q.; Ducheyne, P.; Gao, H.; Ayyaswamy, P.

    1998-01-01

    Using a high aspect ratio vessel (HARV), this study investigated the formation of 3-D rat marrow stromal cell culture on microcarriers and the expression of bone-related biochemical markers under conditions of simulated microgravity. In addition, it calculated the shear stresses imparted on the surface of microcarriers of different densities by the medium fluid in an HARV. Secondary rat marrow stromal cells were cultured on two types of microcarriers, Cytodex-3 beads and modified bioactive glass particles. Examination of cellular morphology by scanning electron microscopy revealed the presence of three-dimensional multicellular aggregates consisting of multiple cell-covered Cytodex-3 microcarriers bridged together. Mineralization was observed in the aggregates. Spherical cell-bead aggregates were observed in an HARV, while cell-bead assemblies were mostly loosely packed in a chain-like or branched structure in a cell bag. The expressions of alkaline phosphatase activity, collagen type I, and osteopontin were shown via the use of histochemical staining, immunolabeling, and confocal scanning electron microscopy. Using a numerical approach, it was found that at a given rotational speed and for a given culture medium, a larger density difference between the microcarrier and the culture medium (e.g., a modified bioactive glass particle) imparted a higher maximum shear stress on the microcarrier.

  4. The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells.

    PubMed

    Yang, Fan; Williams, Christopher G; Wang, Dong-An; Lee, Hyukjin; Manson, Paul N; Elisseeff, Jennifer

    2005-10-01

    Advances in tissue engineering require biofunctional scaffolds that can not only provide cells with structural support, but also interact with cells in a biological manner. To achieve this goal, a frequently used cell adhesion peptide Arg-Gly-Asp (RGD) was covalently incorporated into poly(ethylene glycol) diacrylate (PEODA) hydrogel and its dosage effect (0.025, 1.25 and 2.5 mm) on osteogenesis of marrow stromal cells in a three-dimensional environment was examined. Expression of bone-related markers, osteocalcin (OCN) and Alkaline phosphatase (ALP), increased significantly as the RGD concentration increased. Compared with no RGD, 2.5 mm RGD group showed a 1344% increase in ALP production and a 277% increase in OCN accumulation in the medium. RGD helped MSCs maintain cbfa-1 expression when shifted from a two-dimensional environment to a three-dimensional environment. Soluble RGD was found to completely block the mineralization of marrow stromal cells, as manifested by quantitative calcium assay, phosphorus elemental analysis and Von Kossa staining. In conclusion, we have demonstrated that RGD-conjugated PEODA hydrogel promotes the osteogenesis of MSCs in a dosage-dependent manner, with 2.5 mm being optimal concentration.

  5. Effect of 1 mT sinusoidal electromagnetic fields on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stromal cells.

    PubMed

    Liu, Chaoxu; Yu, Jizhe; Yang, Yong; Tang, Xiangyu; Zhao, Dongming; Zhao, Wenchun; Wu, Hua

    2013-09-01

    Electromagnetic field (EMF) stimulation is clinically beneficial for fracture nonunion and a wide range of bone disorders. However, no consensus has been reached on the optimal parameters of the EMF. The exact mechanism by which EMFs enhance osteogenesis has also not been defined. In the present study, a sinusoidal 1 mT EMF at frequencies of 10, 30, 50, and 70 Hz were administered to rat bone marrow mesenchymal stromal cells (rBMSCs) in the cyclic mode of 2 h exposures followed by 4 h of culture without exposure. The cell viability, proliferation, expression of some osteogenic genes, and mineralization of the extracellular matrix were investigated. It was found that the cell viability was decreased by EMF exposures of 50 and 70 Hz. The proliferation of rBMSCs was elevated significantly in the 10 Hz EMF-treated group during the culture periods. The expression of alkaline phosphatase (ALP) and osteocalcin (OC), two early-phase osteogenic differentiation markers, was up-regulated by the 1 mT, 10 Hz EMF after 1 week. However, the expression of genes that marked the later-phase osteogenic differentiation and maturation of osteoblasts was elevated by the stimulation of 50 Hz EMFs after 2 weeks. In addition, it was observed that the mineralization of the extracellular matrix was enhanced by 50 Hz EMF exposure. These results indicated that the 1 mT EMF at different frequencies had disparate effects on the viability, proliferation and osteogenic differentiation of rBMSCs, and may be beneficial for developing novel therapeutic approaches in bone regenerative medicine. Copyright © 2013 Wiley Periodicals, Inc.

  6. In Vitro Osteogenic Differentiation and In Vivo Bone-Forming Capacity of Human Isogenic Jaw Periosteal Cells and Bone Marrow Stromal Cells

    PubMed Central

    Jaquiéry, Claude; Schaeren, Stefan; Farhadi, Jian; Mainil-Varlet, Pierre; Kunz, Christoph; Zeilhofer, Hans-Florian; Heberer, Michael; Martin, Ivan

    2005-01-01

    Objective: To compare the in vitro osteogenic differentiation and in vivo ectopic bone forming capacity of human bone marrow stromal cells (BMSCs) and jaw periosteal cells (JPCs), and to identify molecular predictors of their osteogenic capacity. Summary Background Data: JPC could be an appealing alternative to BMSC for the engineering of cell-based osteoinductive grafts because of the relatively easy access to tissue with minimal morbidity. However, the extent of osteogenic capacity of JPC has not yet been established or compared with that of BMSC. Methods: BMSCs and JPCs from the same donors (N = 9), expanded for 2 passages, were cultured for 3 weeks in osteogenic medium either in monolayers (Model I) or within 3-dimensional porous ceramic scaffolds, following embedding in fibrin gel (Model II). Cell-fibrin-ceramic constructs were also implanted ectopically in nude mice for 8 weeks (Model III). Cell differentiation in vitro was assessed biochemically and by real-time RT-PCR. Bone formation in vivo was quantified by computerized histomorphometry. Results: JPCs had lower alkaline phosphatase activity, deposited smaller amounts of calcium (Model I), and expressed lower mRNA levels of bone sialoprotein, osteopontin, and osterix (Models I and II) than BMSCs. JPCs produced ectopic bone tissue at lower frequency and amounts (Model III) than BMSCs. Bone sialoprotein, osteopontin, and osterix mRNA levels by BMSCs or JPCs in Model II were markedly higher than in Model I and significantly more expressed by cells that generated bone tissue in Model III. Conclusions: Our data indicate that JPCs, although displaying features of osteogenic cells, would not be as reliable as BMSCs for cell-based bone tissue engineering, and suggest that expression of osteoblast-related markers in vitro could be used to predict whether cells would be osteoinductive in vivo. PMID:16327496

  7. In vitro osteogenic differentiation and in vivo bone-forming capacity of human isogenic jaw periosteal cells and bone marrow stromal cells.

    PubMed

    Jaquiéry, Claude; Schaeren, Stefan; Farhadi, Jian; Mainil-Varlet, Pierre; Kunz, Christoph; Zeilhofer, Hans-Florian; Heberer, Michael; Martin, Ivan

    2005-12-01

    To compare the in vitro osteogenic differentiation and in vivo ectopic bone forming capacity of human bone marrow stromal cells (BMSCs) and jaw periosteal cells (JPCs), and to identify molecular predictors of their osteogenic capacity. JPC could be an appealing alternative to BMSC for the engineering of cell-based osteoinductive grafts because of the relatively easy access to tissue with minimal morbidity. However, the extent of osteogenic capacity of JPC has not yet been established or compared with that of BMSC. BMSCs and JPCs from the same donors (N = 9), expanded for 2 passages, were cultured for 3 weeks in osteogenic medium either in monolayers (Model I) or within 3-dimensional porous ceramic scaffolds, following embedding in fibrin gel (Model II). Cell-fibrin-ceramic constructs were also implanted ectopically in nude mice for 8 weeks (Model III). Cell differentiation in vitro was assessed biochemically and by real-time RT-PCR. Bone formation in vivo was quantified by computerized histomorphometry. JPCs had lower alkaline phosphatase activity, deposited smaller amounts of calcium (Model I), and expressed lower mRNA levels of bone sialoprotein, osteopontin, and osterix (Models I and II) than BMSCs. JPCs produced ectopic bone tissue at lower frequency and amounts (Model III) than BMSCs. Bone sialoprotein, osteopontin, and osterix mRNA levels by BMSCs or JPCs in Model II were markedly higher than in Model I and significantly more expressed by cells that generated bone tissue in Model III. Our data indicate that JPCs, although displaying features of osteogenic cells, would not be as reliable as BMSCs for cell-based bone tissue engineering, and suggest that expression of osteoblast-related markers in vitro could be used to predict whether cells would be osteoinductive in vivo.

  8. The effects of thiazolidinediones on human bone marrow stromal cell differentiation in vitro and in thiazolidinedione-treated patients with type 2 diabetes.

    PubMed

    Beck, George R; Khazai, Natasha B; Bouloux, Gary F; Camalier, Corinne E; Lin, Yiming; Garneys, Laura M; Siqueira, Joselita; Peng, Limin; Pasquel, Francisco; Umpierrez, Denise; Smiley, Dawn; Umpierrez, Guillermo E

    2013-03-01

    Thiazolidinedione (TZD) therapy has been associated with an increased risk of bone fractures. Studies in rodents have led to a model in which decreased bone quality in response to TZDs is due to a competition of lineage commitment between osteoblasts (OBs) and adipocytes (ADs) for a common precursor cell, resulting in decreased OB numbers. Our goal was to investigate the effects of TZD exposure on OB-AD lineage determination from primary human bone marrow stromal cells (hBMSCs) both in vitro and in vivo from nondiabetic subjects and patients with type 2 diabetics. Our experimental design included 2 phases. Phase 1 was an in vitro study of TZD effects on the differentiation of hBMSCs into OBs and ADs in nondiabetic subjects. Phase 2 was a randomized, placebo-controlled trial to determine the effects of 6-month pioglitazone treatment in vivo on hBMSC differentiation using AD/OB colony forming unit assays in patients with type 2 diabetes. In vitro, TZDs (pioglitazone and rosiglitazone) enhanced the adipogenesis of hBMSCs, whereas neither altered OB differentiation or function as measured by alkaline phosphatase activity, gene expression, and mineralization. The ability of TZDs to enhance adipogenesis occurred at a specific time/stage of the differentiation process, and pretreating with TZDs did not further enhance adipogenesis. In vivo, 6-month TZD treatment decreased OB precursors, increased AD precursors, and increased total colony number in patients with type 2 diabetes. Our results indicate that TZD exposure in vitro potently stimulates adipogenesis but does not directly alter OB differentiation/mineralization or lineage commitment from hBMSCs. However, TZD treatment in type 2 diabetic patients results in decreased osteoblastogenesis from hBMSCs compared with placebo, indicating an indirect negative effect on OBs and suggesting an alternative model by which TZDs might negatively regulate bone quality.

  9. Bone morphogenetic protein 2 promotes osteogenesis of bone marrow stromal cells in type 2 diabetic rats via the Wnt signaling pathway.

    PubMed

    Qian, Chao; Zhu, Chenyuan; Yu, Weiqiang; Jiang, Xinquan; Zhang, Fuqiang; Sun, Jian

    2016-11-01

    Type 2 diabetes mellitus impairs osteogenesis in bone marrow stromal cells (BMSCs). Bone morphogenetic protein 2 (BMP2) has been extensively applied for bone defect restoration and has been shown to activate the Wnt signaling pathway. The objective of this study was to investigate the effects of BMP2 on the cell proliferation and osteogenesis of type 2 diabetic BMSCs in rats and explore whether BMP2 induced osteogenesis via the stimulation of Wnt signaling pathway. The cell experiments were divided into DM (diabetic BMSCs), BMP25 (induced with 25ng/ml BMP2), BMP100 (induced with 100ng/ml BMP2) and BMP25 +XAV groups. All cells with or without the different concentrations of BMP2 were cultured under the same experimental conditions. The in vitro results indicated that BMP2 enhanced cell proliferation by 130%-157% and osteogenic differentiation by approximately two-fold in type 2 diabetic BMSCs. The expression levels of β-catenin, cyclin D1, Runx2 and c-myc related to the Wnt signaling pathway were also upregulated from 180% to 212% in BMP2-induced type 2 diabetic rat BMSCs, while the level of GSK3β decreased to 43%. In BMP2-induced type 2 diabetic BMSCs with calcium phosphate cement (CPC) scaffolds for osteoblast study in vivo, the appearance of newly formed bone dramatically increased to 175% compared with type 2 diabetic BMSCs. These data demonstrated that BMP2 enhanced bone regeneration in diabetic BMSCs by stimulating the Wnt signaling pathway with the accumulation of β-catenin and the depressed expression of GSK3β. Diabetic BMSCs associated with BMP2 might be a potential tissue-engineered construct for bone defects in type 2 diabetes mellitus.

  10. The Role of Lipase and α-Amylase in the Degradation of Starch/Poly(ɛ-Caprolactone) Fiber Meshes and the Osteogenic Differentiation of Cultured Marrow Stromal Cells

    PubMed Central

    Martins, Ana M.; Pham, Quynh P.; Malafaya, Patrícia B.; Sousa, Rui A.; Gomes, Manuela E.; Raphael, Robert M.; Kasper, F. Kurtis; Reis, Rui L.

    2009-01-01

    The present work studies the influence of hydrolytic enzymes (α-amylase or lipase) on the degradation of fiber mesh scaffolds based on a blend of starch and poly(ɛ-caprolactone) (SPCL) and the osteogenic differentiation of osteogenic medium–expanded rat bone marrow stromal cells (MSCs) and subsequent formation of extracellular matrix on these scaffolds under static culture conditions. The biodegradation profile of SPCL fiber meshes was investigated using enzymes that are specifically responsible for the enzymatic hydrolysis of SPCL using concentrations similar to those found in human serum. These degradation studies were performed under static and dynamic conditions. After several degradation periods (3, 7, 14, 21, and 30 days), weight loss measurements and micro-computed tomography analysis (specifically porosity, interconnectivity, mean pore size, and fiber thickness) were performed. The SPCL scaffolds were seeded with rat MSCs and cultured for 8 and 16 days using complete osteogenic media with and without enzymes (α-amylase or lipase). Results indicate that culture medium supplemented with enzymes enhanced cell proliferation after 16 days of culture, whereas culture medium without enzymes did not. No calcium was detected in groups cultured with α-amylase or without enzymes after each time period, although groups cultured with lipase presented calcium deposition after the eighth day, showing a significant increase at the sixteenth day. Lipase appears to positively influence osteoblastic differentiation of rat MSCs and to enhance matrix mineralization. Furthermore, scanning electron microscopy images showed that the enzymes did not have a deleterious effect on the three-dimensional structure of SPCL fiber meshes, meaning that the scaffolds did not lose their structural integrity after 16 days. Confocal micrographs have shown cells to be evenly distributed and infiltrated within the SPCL fiber meshes up to 410 μm from the surface. This study demonstrates

  11. Three-dimensional co-culture of mesenchymal stromal cells and differentiated osteoblasts on human bio-derived bone scaffolds supports active multi-lineage hematopoiesis in vitro: Functional implication of the biomimetic HSC niche

    PubMed Central

    Huang, Xiaobing; Zhu, Biao; Wang, Xiaodong; Xiao, Rong; Wang, Chunsen

    2016-01-01

    Recent studies have indicated that the hematopoietic stem/progenitor cell (HSPC) niche, consisting of two major crucial components, namely osteoblasts (OBs) and mesenchymal stromal cells (MSCs), is responsible for the fate of HSPCs. Thus, closely mimicking the HSPC niche ex vivo may be an efficient strategy with which to develop new culture strategies to specifically regulate the balance between HSPC self-renewal and proliferation. The aim of this study was to establish a novel HSPC three-dimensional culture system by co-culturing bone marrow-derived MSCs and OBs differentiated from MSCs without any cytokines as feeder cells and applying bio-derived bone from human femoral metaphyseal portion as the scaffold. Scanning electron microscopy revealed the excellent biocompatibility of bio-derived bone with bone marrow-derived MSCs and OBs differentiated from MSCs. Western blot analysis revealed that many cytokines, which play key roles in HSPC regulation, were comprehensively secreted, while ELISA revealed that extracellular matrix molecules were also highly expressed. Hoechst 33342/propidium iodide fluorescence staining proved that our system could be used to supply a long-term culture of HSPCs. Flow cytometric analysis and qPCR of p21 expression demonstrated that our system significantly promoted the self-renewal and ex vivo expansion of HSPCs. Colony-forming unit (CFU) and long-term culture-initiating cell (LTC-IC) assays confirmed that our system has the ability for both the expansion of CD34+ hematopoietic stem cells (HPCs) and the maintenance of a primitive cell subpopulation of HSCs. The severe-combined immunodeficient mouse repopulating cell assay revealed the promoting effects of our system on the expansion of long-term primitive transplantable HSCs. In conclusion, our system may be a more comprehensive and balanced system which not only promotes the self-renewal and ex vivo expansion of HSPCs, but also maintains primitive HPCs with superior phenotypic and

  12. β-Catenin Directly Sequesters Adipocytic and Insulin Sensitizing Activities but Not Osteoblastic Activity of PPARγ2 in Marrow Mesenchymal Stem Cells

    PubMed Central

    Rahman, Sima; Czernik, Piotr J.; Lu, Yalin; Lecka-Czernik, Beata

    2012-01-01

    Lineage allocation of the marrow mesenchymal stem cells (MSCs) to osteoblasts and adipocytes is dependent on both Wnt signaling and PPARγ2 activity. Activation of PPARγ2, an essential regulator of energy metabolism and insulin sensitivity, stimulates adipocyte and suppresses osteoblast differentiation and bone formation, and correlates with decreased bone mass and increased fracture rate. In contrast, activation of Wnt signaling promotes osteoblast differentiation, augments bone accrual and reduces total body fat. This study examined the cross-talk between PPARγ2 and β-catenin, a key mediator of canonical Wnt signaling, on MSC lineage determination. Rosiglitazone-activated PPARγ2 induced rapid proteolytic degradation of β-catenin, which was prevented by either inhibiting glycogen synthase kinase 3 beta (GSK3β) activity, or blocking pro-adipocytic activity of PPARγ2 using selective antagonist GW9662 or mutation within PPARγ2 protein. Stabilization of β-catenin suppressed PPARγ2 pro-adipocytic but not anti-osteoblastic activity. Moreover, β-catenin stabilization decreased PPARγ2-mediated insulin signaling as measured by insulin receptor and FoxO1 gene expression, and protein levels of phosphorylated Akt (pAkt). Cellular knockdown of β-catenin with siRNA increased expression of adipocyte but did not affect osteoblast gene markers. Interestingly, the expression of Wnt10b was suppressed by anti-osteoblastic, but not by pro-adipocytic activity of PPARγ2. Moreover, β-catenin stabilization in the presence of activated PPARγ2 did not restore Wnt10b expression indicating a dominant role of PPARγ2 in negative regulation of pro-osteoblastic activity of Wnt signaling. In conclusion, β-catenin and PPARγ2 are in cross-talk which results in sequestration of pro-adipocytic and insulin sensitizing activity. The anti-osteoblastic activity of PPARγ2 is independent of this interaction. PMID:23272157

  13. Prostaglandin-mediated inhibition of PTH-stimulated β-catenin signaling in osteoblasts by bone marrow macrophages.

    PubMed

    Estus, Thomas L; Choudhary, Shilpa; Pilbeam, Carol C

    2016-04-01

    Bone marrow macrophages (BMMs), in the presence of cyclooxygenase-2 (Cox2) produced PGE2, secrete an inhibitory factor in response to Rankl that blocks PTH-stimulated osteoblastic differentiation. This study was to determine if the inhibitory factor also blocks PTH-stimulated Wnt signaling. Primary calvarial osteoblasts (POBs) were co-cultured with conditioned medium (CM) from Rankl-treated wild type (WT) BMMs, which make the inhibitory factor, and Cox2 knockout (KO) BMMs, which do not. PTH induced cAMP production was blocked by WT CM but not by KO CM. In the presence of KO CM, PTH induced phosphorylation at β-catenin serine sites, ser552 and ser675, previously shown to be phosphorylated by protein kinase A (PKA). Phosphorylation was blocked by WT CM and by H89, a PKA inhibitor. PTH did not increase total β-catenin. PTH-stimulated transcription factor/lymphoid enhancer-binding factor response element activity in POBs was blocked by WT CM and by serum amyloid A (SAA), the human recombinant analog of murine Saa3, which has recently been shown to be the inhibitory factor. In POBs cultured with Cox2 KO CM, PTH increased expression of multiple genes associated with the anabolic actions of PTH and decreased expression of Wnt antagonists. This differential regulation of gene expression was not seen in POBs cultured with WT CM. These data highlight the ability of PTH to phosphorylate β-catenin directly via PKA and demonstrate the ability of a Cox2-dependent inhibitory factor, secreted by Rankl-stimulated BMMs, to abrogate PTH stimulated β-catenin signaling. Our results suggest that PTH can stimulate a novel negative feedback of its anabolic actions by stimulating Rankl and Cox2 expression. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Prostaglandin-mediated inhibition of PTH-stimulated β-catenin signaling in osteoblasts by bone marrow macrophages

    PubMed Central

    Estus, Thomas L.; Choudhary, Shilpa; Pilbeam, Carol C.

    2016-01-01

    Bone marrow macrophages (BMMs), in the presence of cyclooxygenase-2 (Cox2) produced PGE2, secrete an inhibitory factor in response to Rankl that blocks PTH-stimulated osteoblastic differentiation. This study was to determine if the inhibitory factor also blocks PTH-stimulated Wnt signaling. Primary calvarial osteoblasts (POBs) were co-cultured with conditioned medium (CM) from Rankl-treated wild type (WT) BMMs, which make the inhibitory factor, and Cox2 knockout (KO) BMMs, which do not. PTH induced cAMP production was blocked by WT CM but not by KO CM. In the presence of KO CM, PTH induced phosphorylation at β-catenin serine sites, ser552 and ser675, previously shown to be phosphorylated by protein kinase A (PKA). Phosphorylation was blocked by WT CM and by H89, a PKA inhibitor. PTH did not increase total β-catenin. PTH-stimulated transcription factor/lymphoid enhancer-binding factor response element activity in POBs was blocked by WT CM and by serum amyloid A (SAA), the human recombinant analog of murine Saa3, which has recently been shown to be the inhibitory factor. In POBs cultured with Cox2 KO CM, PTH increased expression of multiple genes associated with the anabolic actions of PTH and decreased expression of Wnt antagonists. This differential regulation of gene expression was not seen in POBs cultured with WT CM. These data highlight the ability of PTH to phosphorylate β-catenin directly via PKA and demonstrate the ability of a Cox2-dependent inhibitory factor, secreted by Rankl-stimulated BMMs, to abrogate PTH stimulated β-catenin signaling. Our results suggest that PTH can stimulate a novel negative feedback of its anabolic actions by stimulating Rankl and Cox2 expression. PMID:26851123

  15. Bone Marrow Stress Decreases Osteogenic Progenitors.

    PubMed

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

    2015-11-01

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

  16. Growth Factors: Production of Monocyte Chemotactic Protein-1 (MCP-1/JE) by Bone Marrow Stromal Cells: Effect on the Migration and Proliferation of Hematopoietic Progenitor Cells.

    PubMed

    Xu, Y. X.; Talati, B. R.; Janakiraman, N.; Chapman, R. A.; Gautam, S. C.

    1999-01-01

    Recombinant chemotactic cytokines (chemokines) have been shown to modulate in vitro proliferation of hematopoietic progenitor cells. Whether bone marrow stromal cells produce chemokines and the physiological role they may have in the regulation of hematopoiesis has largely remained unexamined. We have examined the expression of monocyte chemoattractant protein-1 (MCP-1/JE) in bone marrow stromal cells and its effect on the migration and proliferation of murine hematopoietic progenitor cells. Freshly derived murine bone marrow stromal cells were found to secrete abundant amounts of MCP-1/JE, which was further increased upon stimulation of stromal cells with pro-inflammatory agents LPS, IL1-alpha, IFN-gamma, or TNF-alpha. Although culture supernatant conditioned by stromal cells exhibited chemotactic activity toward hematopoietic progenitor cells, the chemotactic activity was not due to MCP-1/JE. Furthermore, rMCP-1/JE also failed to induce migration of progenitor cells. MCP-1/JE, however, caused 20 to 30% increase in the clonal expansion of progenitor cells. Thus, although MCP-1/JE does not chemoattract hematopoietic progenitor cells it may have a role in their proliferation and clonal expansion.

  17. Manufacturing Differences Affect Human Bone Marrow Stromal Cell Characteristics and Function: Comparison of Production Methods and Products from Multiple Centers

    PubMed Central

    Liu, Shutong; de Castro, Luis F.; Jin, Ping; Civini, Sara; Ren, Jiaqiang; Reems, Jo-Anna; Cancelas, Jose; Nayak, Ramesh; Shaw, Georgina; O’Brien, Timothy; McKenna, David H.; Armant, Myriam; Silberstein, Leslie; Gee, Adrian P.; Hei, Derek J.; Hematti, Peiman; Kuznetsov, Sergei A.; Robey, Pamela G.; Stroncek, David F.

    2017-01-01

    Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression. Among the twenty-four BMSC lots from the eight centers intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from five centers into five distinct clusters. BMSCs from six of the eight centers were tested for their ability to form bone and support hematopoiesis by in vivo transplantation (defining features of BMSCs). Those from all six centers tested formed bone, but the quantity formed was highly variable and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics including their ability to form bone and support hematopoiesis. PMID:28447618

  18. Manufacturing Differences Affect Human Bone Marrow Stromal Cell Characteristics and Function: Comparison of Production Methods and Products from Multiple Centers.

    PubMed

    Liu, Shutong; de Castro, Luis F; Jin, Ping; Civini, Sara; Ren, Jiaqiang; Reems, Jo-Anna; Cancelas, Jose; Nayak, Ramesh; Shaw, Georgina; O'Brien, Timothy; McKenna, David H; Armant, Myriam; Silberstein, Leslie; Gee, Adrian P; Hei, Derek J; Hematti, Peiman; Kuznetsov, Sergei A; Robey, Pamela G; Stroncek, David F

    2017-04-27

    Human bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are manufactured using many different methods, but little is known about the spectrum of manufacturing methods used and their effects on BMSC characteristics and function. Seven centers using, and one developing, Good Manufacturing Practices (GMP) processes were surveyed as to their production methods. Among the seven centers, all used marrow aspirates as the starting material, but no two centers used the same manufacturing methods. Two to four BMSC lots from each center were compared using global gene expression. Among the twenty-four BMSC lots from the eight centers intra-center transcriptome variability was low and similar among centers. Principal component analysis and unsupervised hierarchical clustering analysis separated all the lots from five centers into five distinct clusters. BMSCs from six of the eight centers were tested for their ability to form bone and support hematopoiesis by in vivo transplantation (defining features of BMSCs). Those from all six centers tested formed bone, but the quantity formed was highly variable and BMSCs from only three centers supported hematopoiesis. These results show that differences in manufacturing resulted in variable BMSC characteristics including their ability to form bone and support hematopoiesis.

  19. Requirement of soluble factors produced by bone marrow stromal cells on the growth of novel established human myeloma cell line.

    PubMed

    Aikawa, Shingo; Hatta, Yoshihiro; Tanaka, Megumi; Kaneita, Yoshitaka; Yasukawa, Kiyotaka; Sawada, Umihiko; Horie, Takashi; Tsuboi, Isao; Aizawa, Shin

    2003-03-01

    The growth of myeloma cells is believed to be mediated by functional interactions between tumor cells and the marrow environment involving the action of several cytokines. We report on the establishment and characterization of a new human myeloma cell line (TAB1) that can be long-term maintained in the presence of conditioned medium of bone marrow stromal cells (BMCM) and a BMCM independent variant, C2-2. Both cell lines have plasma cell morphology and express plasma cell antigens (CD38, PCA-1 and immunoglobulin kappa light chain). In the absence of BMCM, TAB1 cells undergoing apoptosis were observed. Among the adherent molecules tested, these cells expressed VLA-4, ICAM-1 and H-CAM, but not VLA-5, suggesting that these were mostly immature plasmacytes. Introduction with exogenous IL-6 and/or GM-CSF, which were detected in BMCM, partially supported the proliferation of TAB1 cells. Treatment with anti-IL-6 antibody partially inhibited the proliferation of TAB1 cells cultured with BMCM. These findings strongly suggest that TAB1 required at least two or more factors on their growth in vitro; IL-6 was one of the factors necessary for cell growth. Further studies are required to clarify the precise molecules which support TAB1 cell growth in combination with IL-6, however, TAB1 and its variant C2-2 cells may offer an attractive model to unravel novel molecular mechanisms involved in bone marrow stroma-dependent growth of myeloma cells.

  20. The cell culture expansion of bone marrow stromal cells from humans with spinal cord injury: implications for future cell transplantation therapy.

    PubMed

    Wright, K T; Masri, W El; Osman, A; Roberts, S; Trivedi, J; Ashton, B A; Johnson, W E B

    2008-12-01

    Previous studies have shown that transplantation of bone marrow stromal cells (MSCs) in animal models of spinal cord injury (SCI) encourages functional recovery. Here, we have examined the growth in cell culture of MSCs isolated from individuals with SCI, compared with non-SCI donors. Centre for Spinal Studies, Midland Centre for Spinal Injuries, RJAH Orthopaedic Hospital, Oswestry, UK. Bone marrow was harvested from the iliac crest of donors with long-term SCI (>3 months, n=9) or from non-SCI donors (n=7). Mononuclear cells were plated out into tissue culture flasks and the adherent MSC population subsequently expanded in monolayer culture. MSC were passaged by trypsinization at 70% confluence and routinely seeded into new flasks at a density of 5 x 10(3) cells per cm(2). Expanded cell cultures were phenotypically characterized by CD-immunoprofiling and by their differentiation potential along chondrocyte, osteoblast and adipocyte lineages. The influence of cell-seeding density on the rate of cell culture expansion and degree of cell senescence was examined in separate experiments. In SCI, but not in non-SCI donors the number of adherent cells harvested at passage I was age-related. The proliferation rate (culture doubling times) between passages I and II was significantly greater in cultures from SCI donors with cervical lesions than in those with thoracic lesions. There was no significant difference, however, in either the overall cell harvests at passages I or II or in the culture doubling times between SCI and non-SCI donors. At passage II, more than 95% of cells were CD34-ve, CD45-ve and CD105+ve, which is characteristic of human MSC cultures. Furthermore, passage II cells differentiated along all three mesenchymal lineages tested. Seeding passage I-III cells at cell densities lower than 5 x 10(3) cells per cm(2) significantly reduced culture doubling times and significantly increased overall cell harvests while having no effect on cell senescence. MSCs from

  1. The role of bone marrow mesenchymal stromal cell derivatives in skin wound healing in diabetic mice

    PubMed Central

    de Mayo, Tomas; Conget, Paulette; Becerra-Bayona, Silvia; Sossa, Claudia L.; Galvis, Virgilio

    2017-01-01

    Mesenchymal stromal cells (MSCs) have shown to be a promising tool in cell therapies to treat different conditions. Several pre-clinical and clinical studies have proved that the transplantation of MSCs improves wound healing. Here, we compare the beneficial effects of mouse bone marrow-derived allogeneic MSCs (allo-mBM-MSCs) and their acelullar derivatives (allo-acd-mMSCs) on skin wound healing in Non-Obese Diabetic (NOD) mice. One dose of allo-mBM-MSCs (1×106 cells) or one dose of allo-acd-mMSCs (1X) were intradermally injected around wounds in 8–10 week old female NOD mice. Wound healing was evaluated macroscopically (wound closure) every two days, and microscopically (reepithelialization, dermoepidermal junction, skin appendage regeneration, leukocyte infiltration, vascularization, granulation tissue formation, and density of collagen fibers in the dermis) after 16 days of MSC injection. In addition, we measured growth factors and specific proteins that were present in the allo-acd-mMSCs. Results showed significant differences in the wound healing kinetics of lesions that received allo-acd-mMSCs compared to lesions that received vehicle or allo-mBM-MSCs. In particular, mice treated with allo-acd-mMSCs reached significantly higher percentages of wound closure at day 4, 6 and 8, relative to the allo-mBM-MSCs and vehicle groups (p < 0.05), while wound closure percentages could not be statistically distinguished between the allo-mBM-MSCs and vehicle groups. Also, allo-acd-mMSCs had a greater influence in the skin would healing process. Specifically, they caused a less pronounced inflammatory severe response (p < 0.0001), more granulation tissue formation at an advanced stage (p < 0.0001), and higher density of collagen fibers (p < 0.05) compared to the other groups. Nevertheless, at day 16, both allo-mBM-MSCs and allo-acd-mMSCs revealed a higher effect on the recovery of the quality skin (continuous epidermis; regular dermoepidermal junction and skin appendages

  2. The role of bone marrow mesenchymal stromal cell derivatives in skin wound healing in diabetic mice.

    PubMed

    de Mayo, Tomas; Conget, Paulette; Becerra-Bayona, Silvia; Sossa, Claudia L; Galvis, Virgilio; Arango-Rodríguez, Martha L

    2017-01-01

    Mesenchymal stromal cells (MSCs) have shown to be a promising tool in cell therapies to treat different conditions. Several pre-clinical and clinical studies have proved that the transplantation of MSCs improves wound healing. Here, we compare the beneficial effects of mouse bone marrow-derived allogeneic MSCs (allo-mBM-MSCs) and their acelullar derivatives (allo-acd-mMSCs) on skin wound healing in Non-Obese Diabetic (NOD) mice. One dose of allo-mBM-MSCs (1×106 cells) or one dose of allo-acd-mMSCs (1X) were intradermally injected around wounds in 8-10 week old female NOD mice. Wound healing was evaluated macroscopically (wound closure) every two days, and microscopically (reepithelialization, dermoepidermal junction, skin appendage regeneration, leukocyte infiltration, vascularization, granulation tissue formation, and density of collagen fibers in the dermis) after 16 days of MSC injection. In addition, we measured growth factors and specific proteins that were present in the allo-acd-mMSCs. Results showed significant differences in the wound healing kinetics of lesions that received allo-acd-mMSCs compared to lesions that received vehicle or allo-mBM-MSCs. In particular, mice treated with allo-acd-mMSCs reached significantly higher percentages of wound closure at day 4, 6 and 8, relative to the allo-mBM-MSCs and vehicle groups (p < 0.05), while wound closure percentages could not be statistically distinguished between the allo-mBM-MSCs and vehicle groups. Also, allo-acd-mMSCs had a greater influence in the skin would healing process. Specifically, they caused a less pronounced inflammatory severe response (p < 0.0001), more granulation tissue formation at an advanced stage (p < 0.0001), and higher density of collagen fibers (p < 0.05) compared to the other groups. Nevertheless, at day 16, both allo-mBM-MSCs and allo-acd-mMSCs revealed a higher effect on the recovery of the quality skin (continuous epidermis; regular dermoepidermal junction and skin appendages

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

    PubMed Central

    Russell, Keith A.; Chow, Natalie H. C.; Dukoff, David; Gibson, Thomas W. G.; LaMarre, Jonathan; Betts, Dean H.; Koch, Thomas G.

    2016-01-01

    Background 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. Hypothesis and Objectives 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. Results 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

  4. Impact of bone marrow stromal cells on Bcl-2 family members in chronic lymphocytic leukemia

    PubMed Central

    Patel, Viralkumar; Balakrishnan, Kumudha; Wierda, William G.; Gandhi, Varsha

    2014-01-01

    Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in the western world. High levels of Bcl-2 family anti-apoptotic proteins are responsible for apoptotic-resistance. Besides anti-apoptotic proteins, microenvironment provides substantial surviving signals to CLL leukemic cells. However, the in-depth-knowledge on the role of individual Bcl-2 family members in the context of microenvironment is still limited. We performed a comprehensive analysis of transcripts and proteins of 18 Bcl-2 family members using “apoptosis array micro fluidic card” in primary cells before and after stromal co-cultures. Our data showed that, 5 of 6 anti-apoptotic members (excluding Bcl-b), 2 of 3 pro-apoptotic members (excluding Bok) and 6 of 9 BH3-only members were present at detectable mRNA levels in CLL cells. Importantly, stromal mediated extended survival of CLL cells was in strong association with elevated global transcription. Upon co-culturing with stromal cells, there was early response of increase in anti- (2/5) and pro-apoptotic protein (3/8) transcripts on day 1, while increase in anti-apoptotic proteins were observed on day 3, with no significant change in pro-apoptotic proteins. Our study revealed a differential pattern of expression of both transcripts and proteins following stromal co-cultures, proposing significance of Bcl-2 family members in stromal microenvironment. PMID:23837491

  5. Osteogenic differentiation of cultured marrow stromal stem cells on surface of microporous hydroxyapatite based mica composite and macroporous synthetic hydroxyapatite.

    PubMed

    Nordström, E; Ohgushi, H; Yoshikawa, T; Yokobori, A T; Yokobori, T

    1999-01-01

    In order to investigate the significance of hydroxyapatite based microporous composite (HA/mica composite) surfaces and a macroporous synthetic hydroxyapatite, rat marrow cell culture, which shows osteogenic differentiation, was carried out on six different culture substrata (two control culture dishes, two identical HA/mica composites, and two identical macroporous synthetic hydroxyapatites). A culture period of two weeks in the presence of beta-glycerophosphate (BGP), ascorbic acid, and dexamethasone resulted in abundant mineralized nodule formations that were positive for alkaline phosphatase (ALP) stain. The stain on the macroporous synthetic hydroxyapatite and the HA/mica composites were intense, the enzyme activity being about double that of control culture dishes. These data indicate that the synthetic macroporous hydroxyapatite surface and the HA/mica composite surface promotes osteoblastic differentiation.

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  7. Safety of treatment with DLA-identical or unrelated mesenchymal stromal cells in DLA-identical canine bone marrow transplantation

    PubMed Central

    Kornblit, Brian; Leisenring, Wendy M.; Santos, Erlinda B.; Storb, Rainer; Sandmaier, Brenda M.

    2013-01-01

    Background: Although in vitro and in vivo experiments have suggested that mesenchymal stromal cells (MSC) may have important immunomodulatory functions in allogeneic hematopoietic cell transplantation (HCT), results from clinical studies have been inconsistent. In the current study we investigate the safety of dog leukocyte antigen (DLA) identical or third party unrelated MSC in DLA-identical HCT. Results: There were no differences between treatment groups in depth of granulocyte or platelet nadirs, time to granulocyte or platelet engraftment, rate of acute GVHD or rejection. All dogs tolerated the MSC infusion well, although 2 dogs treated with unrelated MSC were euthanized on day 9 due to complications unrelated to the MSC infusion. While no formation of ectopic tissue was observed, GFP positive signals in bone marrow, spleen or liver were detected at time of necropsy in 75% and 50% of dogs treated with DLA-identical or unrelated MSC, respectively. Discussion: Treatment with DLA-identical or unrelated MSC in high dose DLA-identical HCT is safe, and provides a large animal HCT model in which to investigate immunological mechanisms and optimal treatment strategies for future human trials. Methods: Fourteen dogs were treated with 920 cGy total body irradiation (TBI) followed by transplantation of marrow from DLA-identical littermates and immunosuppression with cyclosporine. Prior to infusion of marrow, dogs received infusions of DLA-identical MSC from the marrow donor (n = 4), unrelated MSC (n = 4), or culture medium (n = 6), within 1 h of TBI. MSC obtained from relevant donors were ex-vivo expanded and transduced with GFP-retrovirus before infusion. PMID:23723082

  8. Neural and marrow-derived stromal cell sphere transplantation in a rat model of traumatic brain injury.

    PubMed

    Lu, Dunyue; Li, Ya; Mahmood, Asim; Wang, Lei; Rafiq, Tahir; Chopp, Michael

    2002-10-01

    This study was designed to investigate the effect of treatment with a novel composite material consisting of embryonic neurospheres and bone marrow-derived stromal cell spheres (NMSCSs) in a rat model of traumatic brain injury (TBI). The NMSCS composite was injected into the TBI contusion site 24 hours after injury, and all rats were killed on Day 14 after the transplantation. The Rotarod test and the neurological severity score were used to evaluate neurological function. The transplanted NMSCS was analyzed in recipient rat brains by using histological staining and laser scanning confocal microscopy. The lesion volumes in the brains were also calculated using computer image analysis. Rats that received NMSCS transplants had reduced lesion volume and showed improved motor and neurological function when compared with control groups 14 days after the treatment. These results suggest that transplantation of this novel biological material (NMSCS) may be useful in the treatment of TBI.

  9. [Insights into auto-transplantation--the unexpected discovery of transdifferentiation systems in bone marrow stromal cells and its application].

    PubMed

    Dezawa, Mari

    2007-05-01

    Many kinds of cells, including embryonic stem cells and tissue stem cells, have been considered candidates for cell transplantation therapy for muscle-degenerative diseases. Bone marrow stromal cells (MSCs) also have great potential as therapeutic agents since they are easily isolated and can be expanded from patients without serious ethical or technical problems. Recently, new methods for the highly efficient and specific induction of functional skeletal muscle cells have been found in MSCs. Induced cells differentiate into muscle fibers upon transplantation into degenerated muscles of rats and mdx-nude mice. Furthermore, the induced population contained Pax7-positive cells that contribute to subsequent regeneration of muscle upon repetitive damage without additional transplantation of cells. Here I describe the discovery of these induction systems and focus on the potential use of MSC-derived cells for "auto-cell transplantation therapy" in muscle-degenerative diseases.

  10. Potential of Bone Marrow Stromal Cells in Applications for Neuro-Degenerative, Neuro-Traumatic and Muscle Degenerative Diseases

    PubMed Central

    Dezawa, Mari; Ishikawa, Hiroto; Hoshino, Mikio; Itokazu, Yutaka; Nabeshima, Yo-ichi

    2005-01-01

    Cell transplantation is a promising strategy for the treatment of neurodegenerative and muscle degenerative diseases. Many kinds of cells, including embryonic stem cells and tissue stem cells, have been considered as candidates for transplantation therapy. Bone marrow stromal cells (MSCs) have great potential as therapeutic agents since they are easy to isolate and can be expanded from patients without serious ethical or technical problems. We discovered a new method for the highly efficient and specific induction of functional Schwann cells, neurons and skeletal muscle lineage cells from both rat and human MSCs. These induced cells were transplanted into animal models of neurotraumatic injuries, Parkinson’s disease, stroke and muscle dystrophies, resulting in the successful integration of transplanted cells and an improvement in behavior of the transplanted animals. Here we focus on the respective potentials of MSC-derived cells and discuss the possibility of clinical application in degenerative diseases. PMID:18369401

  11. Hydroxyapatite/regenerated silk fibroin scaffold-enhanced osteoinductivity and osteoconductivity of bone marrow-derived mesenchymal stromal cells.

    PubMed

    Jiang, Jia; Hao, Wei; Li, Yuzhuo; Yao, Jinrong; Shao, Zhengzhong; Li, Hong; Yang, Jianjun; Chen, Shiyi

    2013-04-01

    A novel hydroxyapatite/regenerated silk fibroin scaffold was prepared and investigated for its potential to enhance both osteoinductivity and osteoconductivity of bone marrow-derived mesenchymal stromal cells in vitro. Approx. 12.4 ± 0.06 % (w/w) hydroxyapatite was deposited onto the scaffold, and cell viability and DNA content were significantly increased (18.5 ± 0.6 and 33 ± 1.2 %, respectively) compared with the hydroxyapatite scaffold after 14 days. Furthermore, alkaline phosphatase activity in the novel scaffold increased 41 ± 2.5 % after 14 days compared with the hydroxyapatite scaffold. The data indicate that this novel hydroxyapatite/regenerated silk fibroin scaffold has a positive effect on osteoinductivity and osteoconductivity, and may be useful for bone tissue engineering.

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

    SciTech Connect

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

    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.

  13. Cx43 expressed on bone marrow stromal cells plays an essential role in multiple myeloma cell survival and drug resistance

    PubMed Central

    2016-01-01

    Introduction Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is mainly expressed in bone marrow stromal cells (BMSCs) and played a important role on hematopoiesis. In this study, we explored the role of gap junctions (GJs) formed by Cx43 between BMSCs and multiple myeloma (MM) cells. Material and methods qPCR and western blot assays were employed to assay Cx43 expression in three MM cell lines (RPMI 8266, U266, and XG7), freshly isolated MM cells, and bone marrow stromal cells (BMSCs). Cx43 mRNA and proteins were detected in all three MM cell lines and six out of seven freshly isolated MM cells. Resuths The BMSCs from MM patients expressed Cx43 at higher levels than of normal donor (ND-BMSCs). Dye transfer assays demonstrated that gap junction intercellular communication (GJIC) occurring via Cx43 situated between MM and BMSCs is functional. Cytometry beads array (CBA) assays showed that cytokines production changed when the ND-BMSCs were co-cultured with MM cells, especially the levels of IL-6, SDF-1α and IL-10 were higher than those the cells cultured alone and decreased significantly in the presence of GJ inhibitor heptanol. Our results demonstrated that the cytotoxicity of BTZ to MM cells decreased significantly in the presence of BMSCs, an effect that was partially recovered in the presence of GJ inhibitor. Conclusions Our data suggest that GJIC between MM and BMSCs is a critical factor in tumor cell proliferation and drug sensitivity, and is implicated in MM pathogenesis. PMID:28144277

  14. Osterix-cre labeled progenitor cells contribute to the formation and maintenance of the bone marrow stroma.

    PubMed

    Liu, Yaling; Strecker, Sara; Wang, Liping; Kronenberg, Mark S; Wang, Wen; Rowe, David W; Maye, Peter

    2013-01-01

    We have carried out fate mapping studies using Osterix-EGFPCre and Osterix-CreERt animal models and found Cre reporter expression in many different cell types that make up the bone marrow stroma. Constitutive fate mapping resulted in the labeling of different cellular components located throughout the bone marrow, whereas temporal fate mapping at E14.5 resulted in the labeling of cells within a region of the bone marrow. The identity of cell types marked by constitutive and temporal fate mapping included osteoblasts, adipocytes, vascular smooth muscle, perineural, and stromal cells. Prolonged tracing of embryonic precursors labeled at E14.5dpc revealed the continued existence of their progeny up to 10 months of age, suggesting that fate mapped, labeled embryonic precursors gave rise to long lived bone marrow progenitor cells. To provide further evidence for the marking of bone marrow progenitors, bone marrow cultures derived from Osterix-EGFPCre/Ai9 mice showed that stromal cells retained Cre reporter expression and yielded a FACS sorted population that was able to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro and into osteoblasts, adipocytes, and perivascular stromal cells after transplantation. Collectively, our studies reveal the developmental process by which Osterix-Cre labeled embryonic progenitors give rise to adult bone marrow progenitors which establish and maintain the bone marrow stroma.

  15. Osterix-Cre Labeled Progenitor Cells Contribute to the Formation and Maintenance of the Bone Marrow Stroma

    PubMed Central

    Liu, Yaling; Strecker, Sara; Wang, Liping; Kronenberg, Mark S.; Wang, Wen; Rowe, David W.; Maye, Peter

    2013-01-01

    We have carried out fate mapping studies using Osterix-EGFPCre and Osterix-CreERt animal models and found Cre reporter expression in many different cell types that make up the bone marrow stroma. Constitutive fate mapping resulted in the labeling of different cellular components located throughout the bone marrow, whereas temporal fate mapping at E14.5 resulted in the labeling of cells within a region of the bone marrow. The identity of cell types marked by constitutive and temporal fate mapping included osteoblasts, adipocytes, vascular smooth muscle, perineural, and stromal cells. Prolonged tracing of embryonic precursors labeled at E14.5dpc revealed the continued existence of their progeny up to 10 months of age, suggesting that fate mapped, labeled embryonic precursors gave rise to long lived bone marrow progenitor cells. To provide further evidence for the marking of bone marrow progenitors, bone marrow cultures derived from Osterix-EGFPCre/Ai9 mice showed that stromal cells retained Cre reporter expression and yielded a FACS sorted population that was able to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro and into osteoblasts, adipocytes, and perivascular stromal cells after transplantation. Collectively, our studies reveal the developmental process by which Osterix-Cre labeled embryonic progenitors give rise to adult bone marrow progenitors which establish and maintain the bone marrow stroma. PMID:23951132

  16. Evaluation of sericin as a fetal bovine serum-replacing cryoprotectant during freezing of human mesenchymal stromal cells and human osteoblast-like cells.

    PubMed

    Verdanova, Martina; Pytlik, Robert; Kalbacova, Marie Hubalek

    2014-04-01

    A reliable, cryoprotective, xeno-free medium suitable for different cell types is highly desirable in regenerative medicine. There is danger of infection or allergic reaction with the use of fetal bovine serum (FBS), making it problematic for medical applications. The aim of the present study was to develop an FBS-free cryoprotective medium for human mesenchymal stromal cells (hMSCs; primary cells) and immortalized human osteoblasts (SAOS-2 cell line). Furthermore, we endeavored to eliminate or reduce the presence of dimethyl sulfoxide (DMSO) in the medium. Sericin, a sticky protein derived from the silkworm cocoon, was investigated as a substitute for FBS and DMSO in the freezing medium. Cell viability (24 hours after thawing, both hMSC and SAOS-2) and colony-forming ability (2 weeks after thawing, only for hMSCs) were both determined. The FBS-free medium with 1% sericin in 10% DMSO was found to be a suitable freezing medium for primary hMSCs, in contrast to immortalized human osteoblasts. Surprisingly, the storage of hMSCs in a cultivation medium with only 10% DMSO also provided satisfactory results. Any drop in DMSO concentration led to significantly worse survival of cells, with little improvement in hMSC survival in the presence of sericin. Thus, sericin may substitute for FBS in the freezing medium for primary hMSCs, but cannot substitute for DMSO.

  17. [Osteoblastic differentiation and in vivo osteogenic activity of marrow-derived mesenchymal stem cells stimulated by Tacrolimus: experiment with rats].

    PubMed

    Dong, Jian; Dai, Wen-da; Fang, Tao-lin; Lin, Hong; Uemura, Toshimasa

    2007-01-16

    To investigate the effect of the immunosuppressant Tacrolimus (FK506) on the osteoblastic differentiation and in vivo osteogenic inducement of bone marrow-derived mesenchymal stem cells (MSCs). MSCs were derived from Fischer 344 rats. Some MSCs were cultured with L-ascorbic acid-2-phosphate (AsAP) and beta-glycerophosphate (beta-GP) or FK506 plus AsAP and beta-GP. The alkaline phosphatase (APase) activity and calcium deposition were detected 4, 8, 12, and 16 days after the culture. Scanning electron microscopy was used to examine the calcified nodules. Northern blotting was used to detect the mRNA expression of osteocalcium. Multiparous beta-tricalcium phosphate (beta-TCP) ceramic cubes were dipped into 2 kinds of suspension of MSCs, treated by FK506 + AsAP + beta-GP or AsAP + beta-GP, so as to produce 48 pieces of MSCs/beta-TCP complex that were randomly divided into 2 equal groups to be cultured with AsAP + beta-GP or AsAP + beta-GP + FK506 for 4 weeks. The these pieces were transplanted into the subcutaneous sites of the rats' backs and were taken out 4 and 8 weeks later respectively for histological examination. In vitro assays showed that the APase activity, calcium deposition, expression of osteocalcin mRNA of the FK506 + AsAP + beta-GP group at any time points were all significantly higher than those of the AsAP + beta-GP group (all P < 0.05). SEM showed that since the 16th day after culture calcified nodules began to be seen in the FK506 + AsAP + beta-GP group. Since the 4th weeks after transplantation remarkable new bone formation could be seen in the FK506 treated MSCs/beta-TCP complexes in comparison with those MSCs/beta-TCP complexes without treatment with FK506. Greatly enhancing the in vitro osteoblastic differentiation and in vivo osteogenesis of MSCs, FK506 has a potential value as a bone growth factor and may improve the clinical result of bone transplantation used to treat large bone defect. The results of this experiment also contributes to a

  18. Hydrogen gas treatment prolongs replicative lifespan of bone marrow multipotential stromal cells in vitro while preserving differentiation and paracrine potentials.

    PubMed

    Kawasaki, Haruhisa; Guan, Jianjun; Tamama, Kenichi

    2010-07-02

    Cell therapy with bone marrow multipotential stromal cells/mesenchymal stem cells (MSCs) represents a promising approach in the field of regenerative medicine. Low frequency of MSCs in adult bone marrow necessitates ex vivo expansion of MSCs after harvest; however, such a manipulation causes cellular senescence with loss of differentiation, proliferative, and therapeutic potentials of MSCs. Hydrogen molecules have been shown to exert organ protective effects through selective reduction of hydroxyl radicals. As oxidative stress is one of the key insults promoting cell senescence in vivo as well as in vitro, we hypothesized that hydrogen molecules prevent senescent process during MSC expansion. Addition of 3% hydrogen gas enhanced preservation of colony forming early progenitor cells within MSC preparation and prolonged the in vitro replicative lifespan of MSCs without losing differentiation potentials and paracrine capabilities. Interestingly, 3% hydrogen gas treatment did not decrease hydroxyl radical, protein carbonyl, and 8-hydroxydeoxyguanosine, suggesting that scavenging hydroxyl radical might not be responsible for these effects of hydrogen gas in this study.

  19. Platelet lysate favours in vitro expansion of human bone marrow stromal cells for bone and cartilage engineering.

    PubMed

    Zaky, S H; Ottonello, A; Strada, P; Cancedda, R; Mastrogiacomo, M

    2008-12-01

    The heterogeneous population of non-haematopoietic cells residing in the bone marrow (bone marrow stromal cells, BMSCs) and the different fractions and components obtained from platelet-rich plasma provide an invaluable source of autologous cells and growth factors for bone and other connective tissue reconstruction. In this study, we investigated the effect of an allogenic platelet lysate on human BMSCs proliferation and differentiation. Cell proliferation and number of performed cell doublings were enhanced in cultures supplemented with the platelet-derived growth factors (platelet lysate, PL), either with or without the concomitant addition of fetal bovine serum (FBS), compared to cultures performed in the presence of FBS and FGF2. Both in vitro and in vivo osteogenic differentiation were unaltered in cells maintained in medium supplemented with PL and not FBS (Only PL) and in cells maintained in medium containing FBS and FGF2. Interestingly, the in vitro cartilage formation was more effective in the pellet of BMSCs expanded in the Only PL medium. In particular, a chondrogenic differentiation was observed in pellets of some in vitro-expanded BMSCs in the Only PL medium, whereas pellets from parallel cell cultures in medium containing FBS did not respond to the chondrogenic induction. We conclude that the platelet lysate from human source is an effective and even more beneficial substitute for fetal bovine serum to support the in vitro expansion of human BMSCs for subsequent tissue-engineering applications.

  20. Direct reprogramming of human bone marrow stromal cells into functional renal cells using cell-free extracts.

    PubMed

    Papadimou, Evangelia; Morigi, Marina; Iatropoulos, Paraskevas; Xinaris, Christodoulos; Tomasoni, Susanna; Benedetti, Valentina; Longaretti, Lorena; Rota, Cinzia; Todeschini, Marta; Rizzo, Paola; Introna, Martino; Grazia de Simoni, Maria; Remuzzi, Giuseppe; Goligorsky, Michael S; Benigni, Ariela

    2015-04-14

    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.

  1. Immunosuppressive Effects of Multipotent Mesenchymal Stromal Cells on Graft-Versus-Host Disease in Rats Following Allogeneic Bone Marrow Transplantation

    PubMed Central

    Nevruz, Oral; Avcu, Ferit; Ural, A. Uğur; Pekel, Aysel; Dirican, Bahar; Safalı, Mükerrem; Akdağ, Elvin; Beyzadeoğlu, Murat; İde, Tayfun; Sengül, Ali

    2013-01-01

    Objective: Graft-versus-host disease (GVHD) is a major obstacle to successful allogeneic bone marrow transplantation (allo-BMT). While multipotent mesenchymal stromal cells (MSCs) demonstrate alloresponse in vitro and in vivo, they also have clinical applications toward prevention or treatment of GVHD. The aim of this study was to investigate the ability of MSCs to prevent or treat GVHD in a rat BMT model. Materials and Methods: The GVHD model was established by transplantation of Sprague Dawley rats’ bone marrow and spleen cells into lethally irradiated (950 cGy) SDxWistar rat recipients. A total of 49 rats were randomly assigned to 4 study and 3 control groups administered different GVHD prophylactic regimens including MSCs. After transplantation, clinical GVHD scores and survival status were monitored. Results: All irradiated and untreated control mice with GVHD died. MSCs inhibited lethal GVHD as efficiently as the standard GVHD prophylactic regimen. The gross and histopathological findings of GVHD and the ratio of CD4/CD8 expression decreased. The subgroup given MSCs displayed higher in vivo proportions of CD25+ T cells and plasma interleukin-2 levels as compared to conventional GVHD treatment after allo-BMT. Conclusion: Our results suggest that clinical use of MSCs in both prophylaxis against and treatment of established GVHD is effective. This study supports the use of MSCs in the prophylaxis and treatment of GVHD after allo-BMT; however, large scale studies are needed. Conflict of interest:None declared. PMID:24385804

  2. Hydrogen gas treatment prolongs replicative lifespan of bone marrow multipotential stromal cells in vitro while preserving differentiation and paracrine potentials

    SciTech Connect

    Kawasaki, Haruhisa; Guan, Jianjun; Tamama, Kenichi

    2010-07-02

    Cell therapy with bone marrow multipotential stromal cells/mesenchymal stem cells (MSCs) represents a promising approach in the field of regenerative medicine. Low frequency of MSCs in adult bone marrow necessitates ex vivo expansion of MSCs after harvest; however, such a manipulation causes cellular senescence with loss of differentiation, proliferative, and therapeutic potentials of MSCs. Hydrogen molecules have been shown to exert organ protective effects through selective reduction of hydroxyl radicals. As oxidative stress is one of the key insults promoting cell senescence in vivo as well as in vitro, we hypothesized that hydrogen molecules prevent senescent process during MSC expansion. Addition of 3% hydrogen gas enhanced preservation of colony forming early progenitor cells within MSC preparation and prolonged the in vitro replicative lifespan of MSCs without losing differentiation potentials and paracrine capabilities. Interestingly, 3% hydrogen gas treatment did not decrease hydroxyl radical, protein carbonyl, and 8-hydroxydeoxyguanosine, suggesting that scavenging hydroxyl radical might not be responsible for these effects of hydrogen gas in this study.

  3. Marrow-derived stromal cell delivery on fibrin microbeads can correct radiation-induced wound-healing deficits.

    PubMed

    Xie, Michael W; Gorodetsky, Raphael; Micewicz, Ewa D; Micevicz, Ewa D; Mackenzie, Natalia C; Gaberman, Elena; Levdansky, Lilia; McBride, William H

    2013-02-01

    Skin that is exposed to radiation has an impaired ability to heal wounds. This is especially true for whole-body irradiation, where even moderate nonlethal doses can result in wound-healing deficits. Our previous attempts to administer dermal cells locally to wounds to correct radiation-induced deficits were hampered by poor cell retention. Here we improve the outcome by using biodegradable fibrin microbeads (FMBs) to isolate a population of mesenchymal marrow-derived stromal cells (MSCs) from murine bone marrow by their specific binding to the fibrin matrix, culture them to high density in vitro, and deliver them as MSCs on FMBs at the wound site. MSCs are retained locally, proliferate in site, and assist wounds in gaining tensile strength in whole-body irradiated mice with or without additional skin-only exposure. MSC-FMBs were effective in two different mouse strains but were ineffective across a major histocompatability barrier. Remarkably, irradiated mice whose wounds were treated with MSC-FMBs showed enhanced hair regrowth, suggesting indirect effect on the correction of radiation-induced follicular damage. Further studies showed that additional wound-healing benefit could be gained by administration of granulocyte colony-stimulating factor and AMD3100. Collagen strips coated with haptides and MSCs were also highly effective in correcting radiation-induced wound-healing deficits.

  4. [Bone marrow stromal cells. An alternative source of restorative therapy in degenerative diseases of the central nervous system].

    PubMed

    Alberti-Amador, E; García-Miniet, R

    The aim of this study is to describe the capacity of bone marrow cells to limit or slow down the damage and chronic neuronal degeneration produced by degenerative diseases of the central nervous system (CNS), as well as the potential capacity of the method to provide other substances or genetic material. The search for new sources of cells that maintain the ability to divide and distinguish themselves from different cellular phenotypes opens up huge new opportunities in the restorative therapy of these clinical entities. Bone marrow cells, and especially stromal stem cells, have been seen to conserve a high capacity to distinguish and originate different strains of characteristic brain cells (neurons, astrocytes, and glial cells), and also the capacity to restore the population of stem cells when they are stimulated in a suitable fashion. Future experimental studies will be aimed at searching for new ways to enhance the composition, viability and differentiation of the cells to be implanted and will evaluate their effects on diseases of the CNS.

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

    PubMed Central

    Zou, Jixue; Hong, Yan; Tong, Yin; Wei, Ju; Qin, Youwen; Shao, Shan; Wang, Chun; Zhou, Kun

    2015-01-01

    The role of marrow microenvironment in the pathogenesis of myelodysplastic syndrome (MDS) remains controversial. Therefore, we studied the influence of bone marrow-derived mesenchymal stromal cells (BMSCs) from patients with different risk types of MDS on the survival of the MDS cell lines SKM-1 and MUTZ-1. We first demonstrated that the expression of Sonic hedgehog (Shh), smoothened (Smo), and glioma-associated oncogene homolog 1 (Gli1) was increased in MDS patients (n = 23); the increase in expression was positively correlated with the presence of high-risk factors. The Shh signaling inhibitor, cyclopamine, inhibited high-risk MDS BMSC-induced survival of SKM-1 and MUTZ-1 cells, suggesting a role for Shh signaling in MDS cell survival. Furthermore, cyclopamine-mediated inhibition of Shh signaling in SKM-1 and MUTZ-1 cells resulted in decreased DNMT1 expression and cell survival; however, exogenous Shh peptide had the opposite effect, suggesting that Shh signaling could regulate the expression of DNMT1, thereby modulating cell survival in MDS. In addition, the apoptosis of SKM-1 and MUTZ-1 cell increased significantly when cultured with cyclopamine and a demethylation agent, 5-Aza-2′-deoxycytidine. These findings suggest that Shh signaling from BMSCs is important in the pathogenesis of MDS and could play a role in disease progression by modulating methylation. PMID:25861282

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

    PubMed Central

    Papadimou, Evangelia; Morigi, Marina; Iatropoulos, Paraskevas; Xinaris, Christodoulos; Tomasoni, Susanna; Benedetti, Valentina; Longaretti, Lorena; Rota, Cinzia; Todeschini, Marta; Rizzo, Paola; Introna, Martino; Grazia de Simoni, Maria; Remuzzi, Giuseppe; Goligorsky, Michael S.; Benigni, Ariela

    2015-01-01

    Summary 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. PMID:25754206

  7. Transient 100 nM dexamethasone treatment reduces inter- and intraindividual variations in osteoblastic differentiation of bone marrow-derived human mesenchymal stem cells.

    PubMed

    Alm, Jessica J; Heino, Terhi J; Hentunen, Teuvo A; Väänänen, H Kalervo; Aro, Hannu T

    2012-09-01

    The development of in vitro culturing techniques for osteoblastic differentiation of human mesenchymal stem cells (hMSC) is important for cell biology research and the development of tissue-engineering applications. Dexamethasone (Dex) is a commonly used supplement, but the optimal use of Dex treatment is still unclear. By adjusting the timing of Dex supplementation, the negative effects of long-term Dex treatment could be overcome. Transient Dex treatment could contribute toward minimizing broad donor variation, which is a major challenge. We compared the two most widely used Dex concentrations of 10 and 100 nM as transient or continuous treatment and studied inter- and intraindividual variations in osteoblastic differentiation of hMSC. Characterized bone marrow-derived hMSC from 17 female donors of different age groups were used. During osteoblastic induction, the cells were treated with 10 or 100 nM Dex either transiently for different time periods or continuously. Differentiation was evaluated by measuring alkaline phosphatase (ALP) activity and staining for ALP, von Kossa, collagen type I, and osteocalcin. Cell proliferation, cell viability, and apoptosis were also monitored. The strongest osteoblastic differentiation was observed when 100 nM Dex was present for the first week. In terms of inter- and intraindividual coefficients of variations, transient treatment with 100 nM Dex was superior to the other culture conditions and showed the lowest variations in all age groups. This study demonstrates that the temporary presence of 100 nM Dex during the first week of induction culture promotes hMSC osteoblastic differentiation and reduces inter- and intraindividual variations. With this protocol, we can reproducibly produce functional osteoblasts in vitro from the hMSC of different donor populations.

  8. The transcription factors myeloid elf-1-like factor (MEF) and distal-less homeobox 5 (Dlx5) inversely regulate the differentiation of osteoblasts and adipocytes in bone marrow.

    PubMed

    Baek, Kyunghwa; Baek, Jeong-Hwa

    2013-01-01

    In bone marrow, the differentiation of osteoblasts and adipocytes is reciprocally regulated. This inverse regulation occurs mainly through complex signaling crosstalk between transcriptional factors such as peroxisome proliferator-activated receptor-γ (PPARγ) and runt-related transcription factor 2 (Runx2). This commentary addresses the role of myeloid elf-1 like factor (MEF) and distal-less homeobox 5 (Dlx5) in the lineage commitment of bone marrow mesenchymal stem cells into adipocytes and osteoblasts, respectively. MEF suppresses osteoblastogenesis by preventing Runx2 from binding to the promoters of target genes and enhancing adipogenesis via transactivation of PPARγ expression. Conversely, Dlx5 enhances osteoblastogenesis through upregulation of the expression of Runx2 and osteoblast marker genes while suppressing adipogenesis through the downregulation of PPARγ expression by sequestering the cAMP response element binding protein and CCAAT/enhancer-binding protein α. Studies designed to examine the effects of physiological and pathologic signals on the expression of MEF and Dlx5 will provide further insight to the function of these transcription factors in vivo.

  9. Requirement of donor-derived stromal cells in the bone marrow for successful allogeneic bone marrow transplantation. Complete prevention of recurrence of autoimmune diseases in MRL/MP-Ipr/Ipr mice by transplantation of bone marrow plus bones (stromal cells) from the same donor.

    PubMed

    Ishida, T; Inaba, M; Hisha, H; Sugiura, K; Adachi, Y; Nagata, N; Ogawa, R; Good, R A; Ikehara, S

    1994-03-15

    MRL/MP-Ipr/Ipr (MRL/Ipr) mice possess radioresistant (9.5 Gy) abnormal stem cells and show a recurrence of autoimmune diseases within 5 mo of conventional allogeneic bone marrow transplantation. We recently have found that the MHC preference exists between hemopoietic stem cells and stromal cells; when bones are engrafted, donor-derived stromal cells present in the engrafted bones can migrate into the recipient bone marrows, which are replaced with both donor-derived stromal cells and hematopoietic cells. Based on these findings, we attempted to prevent the recurrence of autoimmune diseases in MRL/Ipr mice by the transplantation of both bone marrow cells and bone (as a source of stromal cells). MRL/Ipr mice were irradiated (8.5 Gy) and then reconstituted with C57BL/6 bone marrow cells plus bone grafts. The mice survived more than 48 wk after this treatment. Immunohistologic studies revealed that the mice were completely free from both lymphadenopathy and autoimmune diseases such as lupus nephritis and rheumatoid arthritis. Sera from these mice showed normal levels of circulating immune complexes and rheumatoid factors. Normal functions of both T cells and B cells were noted. Abnormal T cells such as Thy-1+B220+ cells present in nontreated MRL/Ipr mice could not be seen in the thus-treated mice. In addition, to our surprise, spleen cells from treated mice showed completely normal in vitro primary anti-SRBC responses. These results indicate that stromal cells in allogeneic bone marrow transplantation play a crucial role not only in the prevention of graft failure but also in the successful cooperation among APCs, T cells, and B cells. Although MRL/Ipr mice are radiosensitive and usually die of interstitial pneumonia or fatty liver due to the side effects of radiation, it should be noted that this strategy allows a reduction in the radiation dose (9.5 Gy-->8.5 Gy), and that these mice can survive more than 48 wk without showing any symptoms of autoimmune diseases.

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

    PubMed Central

    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

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

    PubMed

    Watt, James; Schlezinger, Jennifer J

    2015-05-04

    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. Chromatin Changes at the PPAR-γ2 Promoter During Bone Marrow-Derived Multipotent Stromal Cell Culture Correlate With Loss of Gene Activation Potential.

    PubMed

    Lynch, Patrick J; Thompson, Elaine E; McGinnis, Kathleen; Rovira Gonzalez, Yazmin I; Lo Surdo, Jessica; Bauer, Steven R; Hursh, Deborah A

    2015-07-01

    Bone marrow-derived multipotent stromal cells (BM-MSCs) display a broad range of therapeutically valuable properties, including the capacity to form skeletal tissues and dampen immune system responses. However, to use BM-MSCs in a clinical setting, amplification is required, which may introduce epigenetic changes that affect biological properties. Here we used chromatin immunoprecipitation to compare post-translationally modified histones at a subset of gene promoters associated with developmental and environmental plasticity in BM-MSCs from multiple donors following culture expansion. At many locations, we observed localization of both transcriptionally permissive (H3K4me3) and repressive (H3K27me3) histone modifications. These chromatin signatures were consistent among BM-MSCs from multiple donors. Since promoter activity depends on the relative levels of H3K4me3 and H3K27me3, we examined the ratio of H3K4me3 to H3K27me3 (K4/K27) at promoters during culture expansion. The H3K4me3 to H3K27me3 ratios were maintained at most assayed promoters over time. The exception was the adipose-tissue specific promoter for the PPAR-γ2 isoform of PPAR-γ, which is a critical positive regulator of adipogenesis. At PPAR-γ2, we observed a change in K4/K27 levels favoring the repressed chromatin state during culture. This change correlated with diminished promoter activity in late passage cells exposed to adipogenic stimuli. In contrast to BM-MSCs and osteoblasts, lineage-restricted preadipocytes exhibited levels of H3K4me3 and H3K27me3 that favored the permissive chromatin state at PPAR-γ2. These results demonstrate that locus-specific changes in H3K4me3 and H3K27me3 levels can occur during BM-MSC culture that may affect their properties. Stem Cells 2015;33:2169-2181. © 2015 AlphaMed Press.

  13. Repair of mandibular defects by bone marrow stromal cells expressing the basic fibroblast growth factor transgene combined with multi-pore mineralized Bio-Oss.

    PubMed

    Yang, Chunyan; Liu, Yang; Li, Chunming; Zhang, Bin

    2013-01-01

    The aim of the present study was to evaluate the effect of combining Bio‑Oss with bone marrow stromal cells (BMSCs) transfected with the basic fibroblast growth factor (bFGF) gene on bone regeneration during mandibular distraction of rabbits. BMSCs obtained from rabbits were transfected with bFGF gene‑encoding plasmids and proliferation rate and the differentiation marker alkaline phosphatase activity were measured. Following seeding into Bio‑Oss collagen and 9‑day culture in vitro, the surface morphology of the Bio‑Oss was assessed using scanning electron microscopy analysis. Three mandibular defects were induced in the lower border of the bilateral mandibular ramus in each New Zealand white rabbit (total n=6). Three scaffolds, group A (seeded with BMSCs/bFGF), B (seeded with BMSCs/pVAX1) and C (cell‑free), which had been cultured in vitro under standard cell culture conditions for 18 days, were implanted into mandibular defects under sterile conditions. Animals were sacrificed by anesthesia overdose 12 weeks following surgery and the scaffolds were extracted for bone mineral density and histological analyses. Results indicate that bFGF was successfully transfected into BMSCs. Proliferation and osteoblast differentiation of BMSCs were stimulated by bFGF in vitro. No differences were identified in surface morphology for Bio‑Oss loaded with variable groups of cells. At week 12 following implantation of Bio‑Oss scaffolds, mineralization of BMSCs in Bio‑Oss scaffolds was observed to be increased by bFGF. New bone and cartilage formation was revealed in hematoxylin and eosin‑stained sections in Bio‑Oss scaffolds and was most abundant in group A (BMSCs transfected with bFGF). In the current study, the bFGF gene was transfected into BMSCs and expressed successfully. bFGF promoted proliferation and differentiation of BMSCs in vitro and implantation of bFGF‑expressing BMSCs combined with Bio‑Oss enhanced new bone regeneration more

  14. Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: enhanced development of mineralized bone-like tissue in culture.

    PubMed

    Pitaru, S; Kotev-Emeth, S; Noff, D; Kaffuler, S; Savion, N

    1993-08-01

    Rat stromal bone marrow cells (SBMC) were shown to produce mineralized bone-like tissue in culture in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate. The addition of 3 ng/ml of basic fibroblast growth factor (bFGF) resulted in a significant increase in formation of mineralized tissue. The present study was aimed at assessing the effect of bFGF on the proliferation and differentiation of SBMC and on the sequential development of mineralized bone-like tissue in culture. Transmission electron microscopy of bFGF-treated cultures demonstrated the development of a multilayered structure resembling mineralized bone tissue consisting of cell layers embedded within a heavy extracellular matrix. The matrix was rich in bundles of collagen fibers associated with extensive mineral deposits consisting of hydroxyapatite as determined by infrared spectrophotometry. The addition of 3 ng/ml of bFGF resulted in significant enhancement of [3H]thymidine and [3H]proline incorporation and protein accumulation by 12-, 2.5-, and 2.5-fold, respectively. bFGF treatment increased cAMP responsiveness, alkaline phosphatase activity, osteocalcin level, 45Ca2+ deposition, and mineralized-like tissue formation and induced the earlier expression of these markers in the treated culture. A biphasic sequence of events was observed during the development of mineralized bone-like tissue in bFGF-treated and control cultures. The first phase is characterized by cell proliferation and matrix accumulation and is reflected by a progressive increase in [3H]thymidine and [3H]proline incorporation until day 11. The second phase, which follows, is characterized by a sharp decline in cell proliferation and matrix accumulation and a concomitant expression of osteoblast differentiation as reflected by the progressive increase in alkaline phosphatase activity, mineral deposition, and osteocalcin expression. Treatment of cultures with bFGF accentuated this biphasic sequence of events. These

  15. Decellularized Iliotibial Band Recolonized with Allogenic Homotopic Fibroblasts or Bone Marrow-Derived Mesenchymal Stromal Cells.

    PubMed

    Gögele, Clemens; Schwarz, Silke; Ondruschka, Benjamin; Hammer, Niels; Schulze-Tanzil, Gundula

    2017-05-10

    Decellularized scaffolds present promising biomimetic approaches in various fields of tissue engineering. Different tissues have been selected for decellularization, among them extracellular matrix (ECM)-rich tissues such as tendons, ligaments and cartilage. The dense ECM of ligaments is particularly challenging to achieve a completely non-immunogenic ECM void of any cells. Here, the methods for decellularization adapted to ligamentous tissue of the iliotibial band (ITB) are presented along with cell isolation and several recolonization techniques using allogenic ITB-derived fibroblasts or mesenchymal stromal cells (MSCs).

  16. Efficient labeling in vitro with non-ionic gadolinium magnetic resonance imaging contrast agent and fluorescent transfection agent in bone marrow stromal cells of neonatal rats

    PubMed Central

    LI, YING-QIN; TANG, YING; FU, RAO; MENG, QIU-HUA; ZHOU, XUE; LING, ZE-MIN; CHENG, XIAO; TIAN, SU-WEI; WANG, GUO-JIE; LIU, XUE-GUO; ZHOU, LI-HUA

    2015-01-01

    Although studies have been undertaken on gadolinium labeling-based molecular imaging in magnetic resonance imaging (MRI), the use of non-ionic gadolinium in the tracking of stem cells remains uncommon. To investigate the efficiency in tracking of stem cells with non-ionic gadolinium as an MRI contrast agent, a rhodamine-conjugated fluorescent reagent was used to label bone marrow stromal cells (BMSCs) of neonatal rats in vitro, and MRI scanning was undertaken. The fluorescent-conjugated cell uptake reagents were able to deliver gadodiamide into BMSCs, and cell uptake was verified using flow cytometry. In addition, the labeled stem cells with paramagnetic contrast medium remained detectable by an MRI monitor for a minimum of 28 days. The present study suggested that this method can be applied efficiently and safely for the labeling and tracking of bone marrow stromal cells in neonatal rats. PMID:25816076

  17. Bone graft in the shape of human mandibular condyle reconstruction via seeding marrow-derived osteoblasts into porous coral in a nude mice model.

    PubMed

    Chen, Fulin; Mao, Tianqiu; Tao, Kai; Chen, Shujun; Ding, Guicong; Gu, Xiaoming

    2002-10-01

    The purpose of this study was to develop a tissue-engineered bone graft model in the shape of a human mandibular condyle. Natural coral with a pore size of 150 to 220 microm and porosity of about 36% was molded into the shape of a human mandibular condyle. Culture-expanded rabbit marrow mesenchymal stem cells were induced by recombinant human bone morphogenetic protein-2 (rhBMP2) to improve osteoblastic phenotype. Then marrow-derived osteoblasts were seeded into natural coral at a density of 2 x 10(8)/mL and incubated in vitro for 3 days before implantation. The cell-coral complexes were implanted subcutaneously into the backs of nude mice and incubated in vivo for 2 months before harvesting. Implantation of coral alone acted as control. The specimens were processed for gross inspection, radiographic examination, and histologic and scanning electronic microscopic observation. The results showed that new bone grafts in the shape of a human mandibular condyle were successfully developed 2 months after implantation and maintained the initial shape of the natural coral scaffold. New bone could be observed histologically on the surface and in the pores of natural coral in all specimens in the cell-seeding group (6 of 6), whereas in the control group there was no evidence of osteogenesis process (0 of 4). This study suggests the feasibility of using porous coral as scaffold material transplanted with marrow-derived osteoblasts to restore bone graft in the shape of human mandibular condyle and shows the potential of using this method for the reconstruction of bone defects. Copyright 2002 American Association of Oral and Maxillofacial Surgeons

  18. Mimicking the functional hematopoietic stem cell niche in vitro: recapitulation of marrow physiology by hydrogel-based three-dimensional cultures of mesenchymal stromal cells.

    PubMed

    Sharma, Monika B; Limaye, Lalita S; Kale, Vaijayanti P

    2012-05-01

    A culture system that closely recapitulates marrow physiology is essential to study the niche-mediated regulation of hematopoietic stem cell fate at a molecular level. We investigated the key features that play a crucial role in the formation of a functional niche in vitro. Hydrogel-based cultures of human placenta-derived mesenchymal stromal cells were established to recapitulate the fibrous three-dimensional architecture of the marrow. Plastic-adherent mesenchymal stromal cells were used as controls. Human bone marrow-derived CD34(+) cells were co-cultured with them. The output hematopoietic cells were characterized by various stem cell-specific phenotypic and functional parameters. The hydrogel-cultures harbored a large pool of primitive hematopoietic stem cells with superior phenotypic and functional attributes. Most importantly, like the situation in vivo, a significant fraction of these cells remained quiescent in the face of a robust multi-lineage hematopoiesis. The retention of a high percentage of primitive stem cells by the hydrogel-cultures was attributed to the presence of CXCR4-SDF1α axis and integrin beta1-mediated adhesive interactions. The hydrogel-grown mesenchymal stromal cells expressed high levels of several molecules that are known to support the maintenance of hematopoietic stem cells. Yet another physiologically relevant property exhibited by the hydrogel cultures was the formation of hypoxia-gradient. Destruction of hypoxia-gradient by incubating these cultures in a hypoxia chamber destroyed their specialized niche properties. Our data show that hydrogel-based cultures of mesenchymal stromal cells form a functional in vitro niche by mimicking key features of marrow physiology.

  19. Functionally and Phenotypically Distinct Subpopulations of Marrow Stromal Cells Are Fibroblast in Origin and Induce Different Fates in Peripheral Blood Monocytes

    PubMed Central

    Iwata, Mineo; Sandstrom, Richard S.; Delrow, Jeffrey J.; Stamatoyannopoulos, John A.

    2014-01-01

    Marrow stromal cells constitute a heterogeneous population of cells, typically isolated after expansion in culture. In vivo, stromal cells often exist in close proximity or in direct contact with monocyte-derived macrophages, yet their interaction with monocytes is largely unexplored. In this report, isolated CD146+ and CD146− stromal cells, as well as immortalized cell lines representative of each (designated HS27a and HS5, respectively), were shown by global DNase I hypersensitive site mapping and principal coordinate analysis to have a lineage association with marrow fibroblasts. Gene expression profiles generated for the CD146+ and CD146− cell lines indicate significant differences in their respective transcriptomes, which translates into differences in secreted factors. Consequently, the conditioned media (CM) from these two populations induce different fates in peripheral blood monocytes. Monocytes incubated in CD146+ CM acquire a tissue macrophage phenotype, whereas monocytes incubated in CM from CD146− cells express markers associated with pre-dendritic cells. Importantly, when CD14+ monocytes are cultured in contact with the CD146+ cells, the combined cell populations, assayed as a unit, show increased levels of transcripts associated with organismal development and hematopoietic regulation. In contrast, the gene expression profile from cocultures of monocytes and CD146− cells does not differ from that obtained when monocytes are cultured with CD146− CM. These in vitro results show that the CD146+ marrow stromal cells together with monocytes increase the expression of genes relevant to hematopoietic regulation. In vivo relevance of these data is suggested by immunohistochemistry of marrow biopsies showing juxtaposed CD146+ cells and CD68+ cells associated with these upregulated proteins. PMID:24131213

  20. Thyrostimulin Regulates Osteoblastic Bone Formation During Early Skeletal Development

    PubMed Central

    van der Spek, Anne; Logan, John G.; Gogakos, Apostolos; Bagchi-Chakraborty, Jayashree; Murphy, Elaine; van Zeijl, Clementine; Down, Jenny; Croucher, Peter I.; Boyde, Alan; Boelen, Anita

    2015-01-01

    The ancestral glycoprotein hormone thyrostimulin is a heterodimer of unique glycoprotein hormone subunit alpha (GPA)2 and glycoprotein hormone subunit beta (GPB)5 subunits with high affinity for the TSH receptor. Transgenic overexpression of GPB5 in mice results in cranial abnormalities, but the role of thyrostimulin in bone remains unknown. We hypothesized that thyrostimulin exerts paracrine actions in bone and determined: 1) GPA2 and GPB5 expression in osteoblasts and osteoclasts, 2) the skeletal consequences of thyrostimulin deficiency in GPB5 knockout (KO) mice, and 3) osteoblast and osteoclast responses to thyrostimulin treatment. Gpa2 and Gpb5 expression was identified in the newborn skeleton but declined rapidly thereafter. GPA2 and GPB5 mRNAs were also expressed in primary osteoblasts and osteoclasts at varying concentrations. Juvenile thyrostimulin-deficient mice had increased bone volume and mineralization as a result of increased osteoblastic bone formation. However, thyrostimulin failed to induce a canonical cAMP response or activate the noncanonical Akt, ERK, or mitogen-activated protein kinase (P38) signaling pathways in primary calvarial or bone marrow stromal cell-derived osteoblasts. Furthermore, thyrostimulin did not directly inhibit osteoblast proliferation, differentiation or mineralization in vitro. These studies identify thyrostimulin as a negative but indirect regulator of osteoblastic bone formation during skeletal development. PMID:26018249

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

    SciTech Connect

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

    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

  2. Phase I trial of repeated intrathecal autologous bone marrow-derived mesenchymal stromal cells in amyotrophic lateral sclerosis.

    PubMed

    Oh, Ki-Wook; Moon, Chanil; Kim, Hyun Young; Oh, Sung-Il; Park, Jinseok; Lee, Jun Ho; Chang, In Young; Kim, Kyung Suk; Kim, Seung Hyun

    2015-06-01

    Stem cell therapy is an emerging alternative therapeutic or disease-modifying strategy for amyotrophic lateral sclerosis (ALS). The aim of this open-label phase I clinical trial was to evaluate the safety of two repeated intrathecal injections of autologous bone marrow (BM)-derived mesenchymal stromal cells (MSCs) in ALS patients. Eight patients with definite or probable ALS were enrolled. After a 3-month lead-in period, autologous MSCs were isolated two times from the BM at an interval of 26 days and were then expanded in vitro for 28 days and suspended in autologous cerebrospinal fluid. Of the 8 patients, 7 received 2 intrathecal injections of autologous MSCs (1 × 10(6) cells per kg) 26 days apart. Clinical or laboratory measurements were recorded to evaluate the safety 12 months after the first MSC injection. The ALS Functional Rating Scale-Revised (ALSFRS-R), the Appel ALS score, and forced vital capacity were used to evaluate the patients' disease status. One patient died before treatment and was withdrawn from the study. With the exception of that patient, no serious adverse events were observed during the 12-month follow-up period. Most of the adverse events were self-limited or subsided after supportive treatment within 4 days. Decline in the ALSFRS-R score was not accelerated during the 6-month follow-up period. Two repeated intrathecal injections of autologous MSCs were safe and feasible throughout the duration of the 12-month follow-up period. Stem cell therapy is an emerging alternative therapeutic or disease-modifying strategy for amyotrophic lateral sclerosis (ALS). To the authors' best knowledge, there are no clinical trials to evaluate the safety of repeated intrathecal injections of autologous bone marrow mesenchymal stromal cells in ALS. After the clinical trial (phase I/II) was conducted, the stem cell (HYNR-CS, NEURONATA-R) was included in the revision of the regulations on orphan drug designation (number 160; December 31, 2013) and approved as

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

    PubMed

    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

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

    PubMed

    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

  5. Bone marrow stromal cells from aged male rats have delayed mineralization and reduced response to mechanical stimulation through nitric oxide and ERK1/2 signaling during osteogenic differentiation.

    PubMed

    Joiner, Danese M; Tayim, Riyad J; Kadado, Allen; Goldstein, Steven A

    2012-10-01

    Bone marrow stromal cells (MSCs) are a source of osteoblast precursors that can be recruited during bone remodeling or injury, both important processes in aging populations. With advancing age, alterations in bone structure and mineralization are often associated with an increase in osteoporosis and fracture risk. Changes in the number of osteoprogenitor cells and their osteogenic potential may occur with advancing age; however few studies have considered the influence of mechanical conditions. Here, we investigated the ability of bone MSCs from mature and aged rats to differentiate into osteoblasts and to respond to short and long periods of mechanical stimulation through signaling by ERK1/2, nitric oxide (NO), and prostaglandin E(2) (PGE(2)) during differentiation. Mineralization was delayed and reduced, but extracellular matrix production appeared less affected by increased age. Differentiating MSCs from aged animals had a decreased response to short and long periods of mechanical stimulation through ERK1/2 signaling, and to long periods of mechanical loading through NO signaling early and late during differentiation. Increases in relative PGE(2) signaling were higher in MSCs from aged animals, which could compensate for reduced ERK1/2 and NO signaling. The decreased mineralization may decrease the ability of cells from aged animals to respond to mechanical stimulation through ERK1/2 and NO signaling, with increased impairment over differentiation time. Decreasing the delay in mineralization of MSCs from aging animals might improve their ability to respond to mechanical stimulation during bone remodeling and injury, suggesting therapies for bone fragility diseases and tissue engineering treatments in elderly populations.

  6. 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.

    PubMed

    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.

  7. Three-dimensional co-culture of mesenchymal stromal cells and differentiated osteoblasts on human bio-derived bone scaffolds supports active multi-lineage hematopoiesis in vitro: Functional implication of the biomimetic HSC niche.

    PubMed

    Huang, Xiaobing; Zhu, Biao; Wang, Xiaodong; Xiao, Rong; Wang, Chunsen

    2016-10-01

    Recent studies have indicated that the hematopoietic stem/progenitor cell (HSPC) niche, consisting of two major crucial components, namely osteoblasts (OBs) and mesenchymal stromal cells (MSCs), is responsible for the fate of HSPCs. Thus, closely mimicking the HSPC niche ex vivo may be an efficient strategy with which to develop new culture strategies to specifically regulate the balance between HSPC self-renewal and proliferation. The aim of this study was to establish a novel HSPC three-dimensional culture system by co-culturing bone marrow-derived MSCs and OBs differentiated from MSCs without any cytokines as feeder cells and applying bio-derived bone from human femoral metaphyseal portion as the scaffold. Scanning electron microscopy revealed the excellent biocompatibility of bio-derived bone with bone marrow-derived MSCs and OBs differentiated from MSCs. Western blot analysis revealed that many cytokines, which play key roles in HSPC regulation, were comprehensively secreted, while ELISA revealed that extracellular matrix molecules were also highly expressed. Hoechst 33342/propidium iodide fluorescence staining proved that our system could be used to supply a long-term culture of HSPCs. Flow cytometric analysis and qPCR of p21 expression demonstrated that our system significantly promoted the self-renewal and ex vivo expansion of HSPCs. Colony-forming unit (CFU) and long-term culture-initiating cell (LTC-IC) assays confirmed that our system has the ability for both the expansion of CD34+ hematopoietic stem cells (HPCs) and the maintenance of a primitive cell subpopulation of HSCs. The severe-combined immunodeficient mouse repopulating cell assay revealed the promoting effects of our system on the expansion of long-term primitive transplantable HSCs. In conclusion, our system may be a more comprehensive and balanced system which not only promotes the self-renewal and ex vivo expansion of HSPCs, but also maintains primitive HPCs with superior

  8. Collagen‐containing scaffolds enhance attachment and proliferation of non‐cultured bone marrow multipotential stromal cells

    PubMed Central

    El‐Jawhari, Jehan J.; Sanjurjo‐Rodríguez, Clara; Jones, Elena

    2015-01-01

    ABSTRACT Large bone defects are ideally treated with autografts, which have many limitations. Therefore, osteoconductive scaffolds loaded with autologous bone marrow (BM) aspirate are increasingly used as alternatives. The purpose of this study was to compare the growth of multipotential stromal cells (MSCs) from unprocessed BM on a collagen‐containing bovine bone scaffold (Orthoss® Collagen) with a non‐collagen‐containing bovine bone scaffold, Orthoss®. Another collagen‐containing synthetic scaffold, Vitoss® was included in the comparison. Colonization of scaffolds by BM MSCs (n = 23 donors) was evaluated using microscopy, colony forming unit‐fibroblast assay and flow‐cytometry. The number of BM MSCs initially attached to Orthoss® Collagen and Vitoss® was similar but greater than Orthoss® (p = 0.001 and p = 0.041, respectively). Furthermore, the number of MSCs released from Orthoss® Collagen and Vitoss® after 2‐week culture was also higher compared to Orthoss® (p = 0.010 and p = 0.023, respectively). Interestingly, collagen‐containing scaffolds accommodated larger numbers of lymphocytic and myelomonocytic cells. Additionally, the proliferation of culture‐expanded MSCs on Orthoss® collagen and Vitoss® was greater compared to Orthoss® (p = 0.047 and p = 0.004, respectively). Collectively, collagen‐containing scaffolds were superior in supporting the attachment and proliferation of MSCs when they were loaded with unprocessed BM aspirates. This highlights the benefit of collagen incorporation into bone scaffolds for use with autologous bone marrow aspirates as autograft substitutes. © 2015 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:597–606, 2016. PMID:26466765

  9. System-wide survey of proteomic responses of human bone marrow stromal cells (hBMSCs) to in vitro cultivation.

    PubMed

    Mindaye, Samuel T; Lo Surdo, Jessica; Bauer, Steven R; Alterman, Michail A

    2015-11-01

    Human bone marrow stromal cells (hBMSCs, also loosely called bone marrow-derived mesenchymal stem cells) are the subject of increasing numbers of clinical trials and laboratory research. Our group recently reported on the optimization of a workflow for a sensitive proteomic study of hBMSCs. Here, we couple this workflow with a label-free protein quantitation method to investigate the molecular responses of hBMSCs to long-term in vitro passaging. We explored the proteomic responses of hBMSCs by assessing the expression levels of proteins at early passage (passage 3, P3) and late passage (P7). We used multiple biological as well as technical replicates to ensure that the detected proteomic changes are repeatable between cultures and thus likely to be biologically relevant. Over 1700 proteins were quantified at three passages and a list of differentially expressed proteins was compiled. Bioinformatics-based network analysis and term enrichment revealed that metabolic pathways are largely altered, where many proteins in the glycolytic, pentose phosphate, and TCA pathways were shown to be largely upregulated in late passages. We also observed significant proteomic alterations in functional categories including apoptosis, and ER-based protein processing and sorting following in vitro cell aging. We posit that the comprehensive map outlined in this report of affected phenotypes as well as the underpinning molecular factors tremendously benefit the effort to uncovering targets that are not just used only to monitor cell fitness but can be employed to slowdown the in vitro aging process in hBMSCs and hence ensure manufacturing of cells with known quality, efficacy and stability.

  10. Collagen-containing scaffolds enhance attachment and proliferation of non-cultured bone marrow multipotential stromal cells.

    PubMed

    El-Jawhari, Jehan J; Sanjurjo-Rodríguez, Clara; Jones, Elena; Giannoudis, Peter V

    2016-04-01

    Large bone defects are ideally treated with autografts, which have many limitations. Therefore, osteoconductive scaffolds loaded with autologous bone marrow (BM) aspirate are increasingly used as alternatives. The purpose of this study was to compare the growth of multipotential stromal cells (MSCs) from unprocessed BM on a collagen-containing bovine bone scaffold (Orthoss(®) Collagen) with a non-collagen-containing bovine bone scaffold, Orthoss(®) . Another collagen-containing synthetic scaffold, Vitoss(®) was included in the comparison. Colonization of scaffolds by BM MSCs (n = 23 donors) was evaluated using microscopy, colony forming unit-fibroblast assay and flow-cytometry. The number of BM MSCs initially attached to Orthoss(®) Collagen and Vitoss(®) was similar but greater than Orthoss(®) (p = 0.001 and p = 0.041, respectively). Furthermore, the number of MSCs released from Orthoss(®) Collagen and Vitoss(®) after 2-week culture was also higher compared to Orthoss(®) (p = 0.010 and p = 0.023, respectively). Interestingly, collagen-containing scaffolds accommodated larger numbers of lymphocytic and myelomonocytic cells. Additionally, the proliferation of culture-expanded MSCs on Orthoss(®) collagen and Vitoss(®) was greater compared to Orthoss(®) (p = 0.047 and p = 0.004, respectively). Collectively, collagen-containing scaffolds were superior in supporting the attachment and proliferation of MSCs when they were loaded with unprocessed BM aspirates. This highlights the benefit of collagen incorporation into bone scaffolds for use with autologous bone marrow aspirates as autograft substitutes.

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

    PubMed

    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.

  12. Bone marrow stromal cells from multiple myeloma patients uniquely induce bortezomib resistant NF-κB activity in myeloma cells

    PubMed Central

    2010-01-01

    Background Components of the microenvironment such as bone marrow stromal cells (BMSCs) are well known to support multiple myeloma (MM) disease progression and resistance to chemotherapy including the proteasome inhibitor bortezomib. However, functional distinctions between BMSCs in MM patients and those in disease-free marrow are not completely understood. We and other investigators have recently reported that NF-κB activity in primary MM cells is largely resistant to the proteasome inhibitor bortezomib, and that further enhancement of NF-κB by BMSCs is similarly resistant to bortezomib and may mediate resistance to this therapy. The mediating factor(s) of this bortezomib-resistant NF-κB activity is induced by BMSCs is not currently understood. Results Here we report that BMSCs specifically derived from MM patients are capable of further activating bortezomib-resistant NF-κB activity in MM cells. This induced activity is mediated by soluble proteinaceous factors secreted by MM BMSCs. Among the multiple factors evaluated, interleukin-8 was secreted by BMSCs from MM patients at significantly higher levels compared to those from non-MM sources, and we found that IL-8 contributes to BMSC-induced NF-κB activity. Conclusions BMSCs from MM patients uniquely enhance constitutive NF-κB activity in MM cells via a proteinaceous secreted factor in part in conjunction with IL-8. Since NF-κB is known to potentiate MM cell survival and confer resistance to drugs including bortezomib, further identification of the NF-κB activating factors produced specifically by MM-derived BMSCs may provide a novel biomarker and/or drug target for the treatment of this commonly fatal disease. PMID:20604947

  13. Vector-averaged gravity regulates gene expression of receptor activator of NF-kappaB (RANK) ligand and osteoprotegerin in bone marrow stromal cells via cyclic AMP/protein kinase A pathway.

    PubMed

    Kanematsu, M; Yoshimura, K; Takaoki, M; Sato, A

    2002-04-01

    Bone loss due to unloading of the skeleton may be caused by an acceleration of osteoclastic bone resorption as well as a decline of osteoblastic bone formation. Recently, two molecular species that play important roles in osteoclastogenesis were discovered: (i) the receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG) ligand/osteoclast differentiation factor induces osteoclastogenesis; and (ii) the OPG/osteoclastogenesis inhibitory factor potently inhibits osteoclastogenesis. To investigate the effects of gravity on gene expression of RANKL and OPG, a mouse bone marrow-derived stromal cell line, ST2, was cultured on a single axis clinostat, which generates a vector-averaged gravity environment. Northern blot analysis revealed that RANKL mRNA was increased, whereas that of OPG decreased. The clinostat culture also caused an increase in intracellular cyclic (cAMP) level. Both forskolin and dibutyryl-cAMP mimicked the regulation of RANKL and OPG transcription in clinostat culture. These modulations of gene expression in clinostat culture were blocked by a protein kinase A (PKA) inhibitor, H89, but not by a cyclooxygenase inhibitor, indomethacin. The enhancement of RANKL gene expression under clinostat culture and its inhibition by H89 were confirmed by a reporter assay with the murine RANKL 5'-flanking region. These results suggest that modulations of RANKL and OPG expression in stromal cells might be one of the causes of bone loss during skeletal unloading. An elevation of intracellular cAMP level caused through an as yet undetermined pathway is involved in modulation of RANKL and OPG expression during clinostat culture.

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

    PubMed

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

    2014-03-01

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

  15. Effective expansion of human adipose-derived stromal cells and bone marrow-derived mesenchymal stem cells cultured on a fragmin/protamine nanoparticles-coated substratum with human platelet-rich plasma.

    PubMed

    Kishimoto, Satoko; Ishihara, Masayuki; Mori, Yasutaka; Takikawa, Megumi; Hattori, Hidemi; Nakamura, Shingo; Sato, Toshinori

    2013-12-01

    Fragmin/protamine nanoparticles (F/P NPs) can be stably coated onto plastic surfaces and used as a substratum for the absorption and controlled release of growth factors (GFs) secreted from human platelet-rich plasma (PRP). In this study, we investigated the capability of F/P NP-coated plates to act as a substratum for the proliferation of human adipose-derived stromal cells (ASCs) and bone marrow-derived mesenchymal stem cells (BMSCs) with GFs in PRP. Both cell types adhered well to the F/P NP-coated plates and grew optimally, with a doubling time of 30 and 32 h in low-concentration PRP (0.5%) medium supplemented with 5 ng/ml fibroblast growth factor-2 (FGF-2) on the F/P NP-coated plates. These cells maintained their multilineage potential for differentiation into adipocytes or osteoblasts. Furthermore, ASCs and BMSCs grew well in medium without PRP and FGF-2 on F/P NP-coated plates pretreated with PRP and FGF-2 in a concentration-dependent manner. Thus, F/P NP-coated plates are a useful substratum for the adherence and proliferation of ASCs and BMSCs in low-concentration PRP medium supplemented with FGF-2. No xenogeneic serum is required. Copyright © 2012 John Wiley & Sons, Ltd.

  16. Human blood and marrow side population stem cell and Stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: correlation with cytokines.

    PubMed

    Brusnahan, S K; McGuire, T R; Jackson, J D; Lane, J T; Garvin, K L; O'Kane, B J; Berger, A M; Tuljapurkar, S R; Kessinger, M A; Sharp, J G

    2010-01-01

    Hematological deficiencies increase with aging leading to anemias, reduced hematopoietic stress responses and myelodysplasias. This study tested the hypothesis that side population hematopoietic stem cells (SP-HSC) would decrease with aging, correlating with IGF-1 and IL-6 levels and increases in bone marrow fat. Marrow was obtained from the femoral head and trochanteric region of the femur at surgery for total hip replacement (N=100). Whole trabecular marrow samples were ground in a sterile mortar and pestle and cellularity and fat content determined. Marrow and blood mononuclear cells were stained with Hoechst dye and the SP-HSC profiles acquired. Marrow stromal cells (MSC) were enumerated flow cytometrically employing the Stro-1 antibody, and clonally in the colony forming unit fibroblast (CFU-F) assay. Plasma levels of IGF-1 (ng/ml) and IL-6 (pg/ml) were measured by ELISA. SP-HSC in blood and bone marrow decreased with age but the quality of the surviving stem cells increased. MSC decreased non-significantly. IGF-1 levels (mean=30.7, SEM=2) decreased and IL-6 levels (mean=4.4, SEM=1) increased with age as did marrow fat (mean=1.2mmfat/g, SEM=0.04). There were no significant correlations between cytokine levels or fat and SP-HSC numbers. Stem cells appear to be progressively lost with aging and only the highest quality stem cells survive.

  17. Human blood and marrow side population stem cell and Stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: Correlation with cytokines

    PubMed Central

    Brusnahan, S.K.; McGuire, T.R.; Jackson, J.D.; Lane, J.T.; Garvin, K.L.; O’Kane, B.J.; Berger, A.M.; Tuljapurkar, S.R.; Kessinger, M.A.; Sharp, J.G.

    2010-01-01

    Hematological deficiencies increase with aging leading to anemias, reduced hematopoietic stress responses and myelodysplasias. This study tested the hypothesis that side population hematopoietic stem cells (SP-HSC) would decrease with aging, correlating with IGF-1 and IL-6 levels and increases in bone marrow fat. Marrow was obtained from the femoral head and trochanteric region of the femur at surgery for total hip replacement (N = 100). Whole trabecular marrow samples were ground in a sterile mortar and pestle and cellularity and fat content determined. Marrow and blood mononuclear cells were stained with Hoechst dye and the SP-HSC profiles acquired. Marrow stromal cells (MSC) were enumerated flow cytometrically employing the Stro-1 antibody, and clonally in the colony forming unit fibroblast (CFU-F) assay. Plasma levels of IGF-1 (ng/ml) and IL-6 (pg/ml) were measured by ELISA. SP-HSC in blood and bone marrow decreased with age but the quality of the surviving stem cells increased. MSC decreased non-significantly. IGF-1 levels (mean = 30.7, SEM = 2) decreased and IL-6 levels (mean = 4.4, SEM = 1) increased with age as did marrow fat (mean = 1.2 mm fat/g, SEM = 0.04). There were no significant correlations between cytokine levels or fat and SP-HSC numbers. Stem cells appear to be progressively lost with aging and only the highest quality stem cells survive. PMID:21035480

  18. Migration of bone marrow stromal cells in 3D: 4 color methodology reveals spatially and temporally coordinated events.

    PubMed

    Thibault, Marc M; Buschmann, Michael D

    2006-12-01

    The cytoskeleton plays a central role in many cell processes including directed cell migration. Since most previous work has investigated cell migration in two dimensions (2D), new methods are required to study movement in three dimensions (3D) while preserving 3D structure of the cytoskeleton. Most previous studies have labeled two cytoskeletal networks simultaneously, impeding an appreciation of their complex and dynamic interconnections. Here we report the development of a 4 color method to simultaneously image vimentin, actin, tubulin and the nucleus for high-resolution confocal microscopy of bone-marrow stromal cells (BMSCs) migrating through a porous membrane. Several methods were tested for structural preservation and labeling intensity resulting in identification of an optimized simultaneous fixation and permeabilization method using glutaraldehyde, paraformaldehyde and Triton X-100 followed by a quadruple fluorescent labeling method. This procedure was then applied at a sequence of time points to migrating cells, allowing temporal progression of migration to be assessed by visualizing all three networks plus the nucleus, providing new insights into 3D directed cell migration including processes such as leading edge structure, cytoskeletal distribution and nucleokinesis. Colocalization of actin and microtubules with distinct spatial arrangements at the cellular leading edge during migration, together with microtubule axial polarization supports recent reports indicating the pivotal role of microtubules in directed cell migration. This study also provides a foundation for 3D migration studies versus 2D studies, providing precise and robust methods to attain new insights into the cellular mechanisms of motility.

  19. Bone Marrow Stromal Cell Intraspinal Transplants Fail to Improve Motor Outcomes in a Severe Model of Spinal Cord Injury

    PubMed Central

    Brock, John H.; Graham, Lori; Staufenberg, Eileen; Collyer, Eileen; Koffler, Jacob

    2016-01-01

    Abstract Bone marrow stromal cells (BMSCs) have been reported to exert potential neuroprotective properties in models of neurotrauma, although precise mechanisms underlying their benefits are poorly understood. Despite this lack of knowledge, several clinical trials have been initiated using these cells. To determine whether local mechanisms mediate BMSC neuroprotective actions, we grafted allogeneic BMSCs to sites of severe, compressive spinal cord injury (SCI) in Sprague-Dawley rats. Cells were administered 48 h after the original injury. Additional animals received allogeneic MSCs that were genetically modified to secrete brain-derived neurotrophic factor (BDNF) to further determine whether a locally administered neurotrophic factor provides or extends neuroprotection. When assessed 2 months post-injury in a clinically relevant model of severe SCI, BMSC grafts with or without BDNF secretion failed to improve motor outcomes. Thus, allogeneic grafts of BMSCs do not appear to act through local mechanisms, and future clinical trials that acutely deliver BMSCs to actual sites of injury within days are unlikely to be beneficial. Additional studies should address whether systemic administration of BMSCs alter outcomes from neurotrauma. PMID:26414795

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Transplanted bone marrow stromal cells protect neurovascular units and ameliorate brain damage in stroke-prone spontaneously hypertensive rats.

    PubMed

    Ito, Masaki; Kuroda, Satoshi; Sugiyama, Taku; Maruichi, Katsuhiko; Kawabori, Masahito; Nakayama, Naoki; Houkin, Kiyohiro; Iwasaki, Yoshinobu

    2012-10-01

    This study was aimed to assess whether bone marrow stromal cells (BMSC) could ameliorate brain damage when transplanted into the brain of stroke-prone spontaneously hypertensive rats (SHR-SP). The BMSC or vehicle was stereotactically engrafted into the striatum of male SHR-SP at 8 weeks of age. Daily loading with 0.5% NaCl-containing water was started from 9 weeks. MRIs and histological analysis were performed at 11 and 12 weeks, respectively. Wistar-Kyoto rats were employed as the control. As a result, T2-weighted images demonstrated neither cerebral infarct nor intracerebral hemorrhage, but identified abnormal dilatation of the lateral ventricles in SHR-SP. HE staining demonstrated selective neuronal injury in their neocortices. Double fluorescence immunohistochemistry revealed that they had a decreased density of the collagen IV-positive microvessels and a decreased number of the microvessels with normal integrity between basement membrane and astrocyte end-feet. BMSC transplantation significantly ameliorated the ventricular dilatation and the breakdown of neurovascular integrity. These findings strongly suggest that long-lasting hypertension may primarily damage neurovascular integrity and neurons, leading to tissue atrophy and ventricular dilatation prior to the occurrence of cerebral stroke. The BMSC may ameliorate these damaging processes when directly transplanted into the brain, opening the possibility of prophylactic medicine to prevent microvascular and parenchymal-damaging processes in hypertensive patients at higher risk for cerebral stroke.

  2. Connective tissue growth factor regulates adipocyte differentiation of mesenchymal stromal cells and facilitates leukemia bone marrow engraftment

    PubMed Central

    Battula, V. Lokesh; Chen, Ye; Cabreira, Maria da Graca; Ruvolo, Vivian; Wang, Zhiqiang; Ma, Wencai; Konoplev, Sergej; Shpall, Elizabeth; Lyons, Karen; Strunk, Dirk; Bueso-Ramos, Carlos; Davis, Richard Eric; Konopleva, Marina

    2013-01-01

    Mesenchymal stromal cells (MSCs) are a major component of the leukemia bone marrow (BM) microenvironment. Connective tissue growth factor (CTGF) is highly expressed in MSCs, but its role in the BM stroma is unknown. Therefore, we knocked down (KD) CTGF expression in human BM-derived MSCs by CTGF short hairpin RNA. CTGF KD MSCs exhibited fivefold lower proliferation compared with control MSCs and had markedly fewer S-phase cells. CTGF KD MSCs differentiated into adipocytes at a sixfold higher rate than controls in vitro and in vivo. To study the effect of CTGF on engraftment of leukemia cells into BM, an in vivo model of humanized extramedullary BM (EXM-BM) was developed in NOD/SCID/IL-2rgnull mice. Transplanted Nalm-6 or Molm-13 human leukemia cells engrafted at a threefold higher rate in adipocyte-rich CTGF KD MSC-derived EXM-BM than in control EXM-BM. Leptin was found to be highly expressed in CTGF KD EXM-BM and in BM samples of patients with acute myeloid and acute lymphoblastic leukemia, whereas it was not expressed in normal controls. Given the established role of the leptin receptor in leukemia cells, the data suggest an important role of CTGF in MSC differentiation into adipocytes and of leptin in homing and progression of leukemia. PMID:23741006

  3. Transplantation of NGF-gene-modified bone marrow stromal cells into a rat model of Alzheimer' disease.

    PubMed

    Li, Li-Yan; Li, Jin-Tao; Wu, Qing-Ying; Li, Jin; Feng, Zhong-Tang; Liu, Su; Wang, Ting-Hua

    2008-02-01

    It is well known that bone marrow stromal cells (BMSC) grafted into the hippocampus of the rat model of Alzheimer's disease (AD) could survive and differentiate into cholinergic neurons as well as contribute towards functional restoration. The present study evaluated the effects of BMSC as a seed cell modified by nerve growth factor (NGF) gene into the hippocampus of AD rats. The beta-amyloid protein was injected bilaterally into the rat hippocampus to reproduce the AD model. After the human total RNA was extracted, the NGF gene was amplified by reverse transcription-polymerase chain reaction, then cloned into the pcDNA3. BMSC derived from a green fluorescence protein transgenic mouse were isolated, cultured, identified, and transfected by the NGF recombinant. The NGF-gene-modified BMSC were then transplanted into the hippocampus of AD rats. The results showed that implanted BMSC survived, migrated and expressed NGF as well as differentiated into ChAT-positive neurons. A significant improvement in learning and memory in AD rats was also seen in NGF-gene-modified BMSC group, when compared with the BMSC group. The present findings suggested that BMSC provided an effective carrier for delivery of NGF into AD rats, and the administration of NGF-gene-modified BMSC may be considered as a potential strategy for the development of effective therapies for the treatment of AD.

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

    PubMed

    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.

  5. Synergistic effects of ultrashort wave and bone marrow stromal cells on nerve regeneration with acellular nerve allografts.

    PubMed

    Pang, Chao-Jian; Tong, Lei; Ji, Li-Li; Wang, Zhen-Yu; Zhang, Xu; Gao, Hai; Jia, Hua; Zhang, Li-Xin; Tong, Xiao-Jie

    2013-10-01

    Acellular nerve allografts (ANA) possess bioactivity and neurite promoting factors in nerve tissue engineering. Previously we reported that low dose ultrashort wave (USW) radiation could enhance the rate and quality of peripheral nerve regeneration with ANA repairing sciatic nerve defects. Meanwhile, ANA implanted with bone marrow stromal cells (BMSCs) exhibited a similar result. Thus, it is interesting to know whether it might yield a synergistic effect when USW radiation is combined with BMSCs-laden ANA. Here we investigated the effectiveness of ANA seeded with BMSCs, combined with USW therapy on repairing peripheral nerve injuries. Adult male Wistar rats were randomly divided into four groups: Dulbecco's modified Eagle's medium (DMEM) control group, BMSCs-laden group, ultrashort wave (USW) group and BMSC + USW group. The regenerated nerves were assayed morphologically and functionally, and growth-promoting factors in the regenerated tissues following USW administration or BMSCs integration were also detected. The results indicated that the combination therapy caused much better beneficial effects evidenced by increased myelinated nerve fiber number, myelin sheath thickness, axon diameter, sciatic function index, nerve conduction velocity, and restoration rate of tibialis anterior wet weight. Moreover, the mRNA levels of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in the spinal cord and muscles were elevated significantly. In conclusion, we found a synergistic effect of USW radiation and BMSCs treatment on peripheral nerve regeneration, which may help establish novel strategies for repairing peripheral nerve defects. Copyright © 2013 Wiley Periodicals, Inc.

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

    SciTech Connect

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

    2014-10-24

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

  7. Scaffold-free and scaffold-assisted 3D culture enhances differentiation of bone marrow stromal cells.

    PubMed

    Vidyasekar, Prasanna; Shyamsunder, Pavithra; Sahoo, Sanjeeb Kumar; Verma, Rama Shanker

    2016-02-01

    3D cultures of stem cells can preserve differentiation potential or increase the efficiency of methods that induce differentiation. Mouse bone marrow-derived stromal cells (BMSCs) were cultured in 3D as scaffold-free spheroids or "mesoid bodies" (MBs) and as aggregates on poly(lactic) acid microspheres (MB/MS). 3D cultures demonstrated viable cells, interaction on multiple planes, altered cell morphology, and the formation of structures similar to epithelial cell bridges. Cell proliferation was limited in suspension cultures of MB and MB/MS; however, cells regained proliferative capacity when transferred to flat substrates of tissue culture plates (TCPs). Expanded as monolayer, cells retained expression of Sca-1 and CD44 stem cell markers. 3D cultures demonstrated enhanced potential for adipogenic and osteogenic differentiation showing higher triglyceride accumulation and robust mineralization in comparison with TCP cultures. Enhanced and efficient adipogenesis was also observed in 3D cultures generated in a rotating cell culture system. Preservation of multilineage potential of BMSC was demonstrated in 5-azacytidine treatment of 3D cultures and TCP by expression of cardiac markers GATA4 and ACTA1 although functioning cardiomyocytes were not derived.

  8. Low-intensity pulsed ultrasound (LIPUS) and cell-to-cell communication in bone marrow stromal cells.

    PubMed

    Sena, Kotaro; Angle, Siddhesh R; Kanaji, Arihiko; Aher, Chetan; Karwo, David G; Sumner, Dale R; Virdi, Amarjit S

    2011-07-01

    Low-intensity pulsed ultrasound (LIPUS) is an established therapy for fracture repair and has been used widely in the clinics, but its underlying mechanism of action remains unclear. The aim of the current research was to determine the effect of LIPUS on gap junctional cell-to-cell intercellular communication in rat bone marrow stromal cells (BMSC) in vitro and to determine whether the ability of BMSCs to communicate by gap junctions would affect their response to LIPUS. Single or daily-multiple LIPUS treatment at 1.5MHz, 30mW/cm(2), for 20min was applied to BMSC. We demonstrated that BMSC form functional gap junctions and single LIPUS treatment significantly increased the intracellular dye transfer between BMSC. In addition, activated phosphorylation of ERK1/2 and p38 by LIPUS stimulation was diminished when cells were treated with a gap junction inhibitor 18β-glycyrrhetinic acid (18β). We further demonstrated that 18β diminished the significant increase in alkaline phosphatase activity following LIPUS stimulation. These results suggest a potential role of gap junctional cell-to-cell intercellular communication on the effects of LIPUS in BMSC.

  9. Molecular targeting regulation of proliferation and differentiation of the bone marrow-derived mesenchymal stem cells or mesenchymal stromal cells.

    PubMed

    Chen, Bei-Yu; Wang, Xi; Chen, Liang-Wei; Luo, Zhuo-Jing

    2012-04-01

    The bone marrow-derived mesenchymal stem cells or mesenchymal stromal cells (MSCs), with pluripotent differentiation capacity, present an ideal source for cell transplantation or tissue engineering therapies, but exact understanding of regulating mechanism underling MSC proliferation and differentiation remains a critical issue in securing their safe and efficient clinical application. This review outlines current knowledge regarding MSC cell surface biomarkers and molecular mechanisms of MSC differentiation and proliferation with emphasis on Wnt/β-catenin signaling, Notch signaling pathway, bone morphogenesis proteins and various growth factors functioning in regulation of differentiation and proliferation of MSCs. Possible relation of oncogene and immunosuppressive activities of MSCs with tumorigenicity or tumor generation is also addressed for safe translational clinical application. Fast increase of MSC knowledge and techniques has led to some successful clinical trials and helped devising new tissue engineering therapies for bone and cartilage diseases that severely afflict human health. Production of adult MSC-derived functional neurons can further extend their therapeutic application in nerve injury and neurodegenerative diseases. It is promising that MSCs shall overcome ethical and immunorejection problems appeared in human embryonic stem cells, and specific molecular targeting manipulation may result in practical MSC therapy for personalized treatment of various diseases in the regeneration medicine.

  10. Expansion induced microRNA changes in bone marrow mesenchymal stromal cells reveals interplay between immune regulation and cell cycle

    PubMed Central

    Kilpinen, Lotta; Parmar, Amarjit; Greco, Dario; Korhonen, Matti; Lehenkari, Petri; Saavalainen, Päivi; Laitinen, Saara

    2016-01-01

    Mesenchymal stromal cells (MSC) are currently used in many cell based therapies. Prior to use in therapy, extensive expansion is required. We used microarray profiling to investigate expansion induced miRNA and mRNA expression changes of bone marrow MSCs (BM-MSCs) derived from old and young donors. The expression levels of 36 miRNAs were altered in cells derived from the old and respectively 39 miRNAs were altered in cells derived from young donors. Of these, only 12 were differentially expressed in both young and old donor BM-MSCs, and their predicted target mRNAs, were mainly linked to cell proliferation and senescence. Further qPCR verification showed that the expression of miR-1915-3p, miR-1207, miR-3665, and miR-762 correlated with the expansion time at passage 8. Previously described BM-MSC-specific miRNA fingerprints were also detected but these remained unchanged during expansion. Interestingly, members of well-studied miR-17/92 cluster, involved in cell cycle regulation, aging and also development of immune system, were down-regulated specifically in cells from old donors. The role of this cluster in MSC functionality is worth future studies since it links expansion, aging and immune system together. PMID:27852979

  11. Tungsten Promotes Sex-Specific Adipogenesis in the Bone by Altering Differentiation of Bone Marrow-Resident Mesenchymal Stromal Cells.

    PubMed

    Bolt, Alicia M; Grant, Michael P; Wu, Ting Hua; Flores Molina, Manuel; Plourde, Dany; Kelly, Alexander D R; Negro Silva, Luis Fernando; Lemaire, Maryse; Schlezinger, Jennifer J; Mwale, Fackson; Mann, Koren K

    2016-04-01

    Tungsten is a naturally occurring metal that increasingly is being incorporated into industrial goods and medical devices, and is recognized as an emerging contaminant. Tungsten preferentially and rapidly accumulates in murine bone in a concentration-dependent manner; however the effect of tungsten deposition on bone biology is unknown. Other metals alter bone homeostasis by targeting bone marrow-derived mesenchymal stromal cell (MSC) differentiation, thus, we investigated the effects of tungsten on MSCsin vitroandin vivoIn vitro, tungsten shifted the balance of MSC differentiation by enhancing rosiglitazone-induced adipogenesis, which correlated with an increase in adipocyte content in the bone of tungsten-exposed, young, male mice. Conversely, tungsten inhibited osteogenesis of MSCsin vitro; however, we found no evidence that tungsten inhibited osteogenesisin vivo Interestingly, two factors known to influence adipogenesis are sex and age of mice. Both female and older mice have enhanced adipogenesis. We extended our study and exposed young female and adult (9-month) male and female mice to tungsten for 4 weeks. Although tungsten accumulated to a similar extent in young female mice, it did not promote adipogenesis. Interestingly, tungsten did not accumulate in the bone of older mice; it was undetectable in adult male mice, and just above the limit of detect in adult female mice. Surprisingly, tungsten enhanced adipogenesis in adult female mice. In summary, we found that tungsten alters bone homeostasis by altering differentiation of MSCs, which could have significant implications for bone quality, but is highly dependent upon sex and age.

  12. Proliferation and osteogenic differentiation of human bone marrow stromal cells on alginate-gelatine-hydroxyapatite scaffolds with anisotropic pore structure.

    PubMed

    Bernhardt, A; Despang, F; Lode, A; Demmler, A; Hanke, T; Gelinsky, M

    2009-01-01

    Porous mineralized scaffolds are required for various applications in bone engineering. In particular, tube-like pores with controlled orientation inside the scaffold may support homogeneous cell seeding as well as sufficient nutrient supply and may facilitate blood vessel ingrowth. Scaffolds with parallely orientated tube-like pores were generated by diffusion-controlled ionotropic gelation of alginate. Incorporation of hydroxyapatite (HA) during the gelation process yielded stable scaffolds with an average pore diameter of approximately 90 microm. To evaluate the potential use of alginate-gelatine-HA scaffolds for bone tissue engineering, in vitro tests with human bone marrow stromal cells (hBMSCs) were carried out. We analysed biocompatibility and cell penetration into the capillary pores by microscopic methods. hBMSCs were also cultivated on alginate-gelatine-HA scaffolds for 3 weeks in the presence and absence of osteogenic supplements. We studied proliferation and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content and alkaline phosphatase (ALP) activity and found a 10-14-fold increase of cell number after 2 weeks of cultivation, as well as an increase of specific ALP activity for osteogenic-induced hBMSCs. Furthermore, the expression of bone-related genes [ALP, bone sialoprotein II (BSPII)] was analysed. We found an increase of ALP as well as BSPII expression for osteogenic-induced hBMSCs on alginate-gelatin-HA scaffolds. 2008 John Wiley & Sons, Ltd

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

    PubMed

    Ide, Chizuka; Nakano, Norihiko; Kanekiyo, Kenji

    2016-09-01

    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.

  14. Bone marrow stromal cell-mediated tissue sparing enhances functional repair after spinal cord contusion in adult rats.

    PubMed

    Ritfeld, Gaby J; Nandoe Tewarie, Rishi D S; Vajn, Katarina; Rahiem, Sahar T; Hurtado, Andres; Wendell, Dane F; Roos, Raymund A C; Oudega, Martin

    2012-01-01

    Bone marrow stromal cell (BMSC) transplantation has shown promise for repair of the spinal cord. We showed earlier that a BMSC transplant limits the loss of spinal nervous tissue after a contusive injury. Here, we addressed the premise that BMSC-mediated tissue sparing underlies functional recovery in adult rats after a contusion of the thoracic spinal cord. Our results reveal that after 2 months BMSCs had elicited a significant increase in spared tissue volumes and in blood vessel density in the contusion epicenter. A strong functional relationship existed between spared tissue volumes and blood vessel density. BMSC-transplanted rats exhibited significant improvements in motor, sensorimotor, and sensory functions, which were strongly correlated with spared tissue volumes. Retrograde tracing revealed that rats with BMSCs had twice as many descending brainstem neurons with an axon projecting beyond the contused spinal cord segment and these correlated strongly with the improved motor/sensorimotor functions but not sensory functions. Together, our data indicate that tissue sparing greatly contributes to BMSC-mediated functional repair after spinal cord contusion. The preservation/formation of blood vessels and sparing/regeneration of descending brainstem axons may be important mediators of the BMSC-mediated anatomical and functional improvements.

  15. Perilesional intrathecal administration of autologous bone marrow stromal cells achieves functional improvement in pigs with chronic paraplegia.

    PubMed

    Zurita, Mercedes; Aguayo, Concepcion; Bonilla, Celia; Rodriguez, Alicia; Vaquero, Jesus

    2013-10-01

    At present, on the basis of the great number of preclinical studies and preliminary clinical trials in humans, bone marrow stromal cell (BMSC) transplantation offers promise in the treatment of paraplegia. Nevertheless, there is not enough experience in humans about the best candidates for this type of cell therapy or details about the best parameters or best route of administration. Two adult paraplegic pigs with chronic paraplegia were treated only with perilesional intrathecal administration of 40 × 10(6) autologous BMSC suspended in autologous plasma and followed for 1 year after cell transplantation. Our study showed clinical improvement, starting at 2 mo after BMSC administration and reaching stabilization at 10 mo. This was associated with recovery of previously abolished somatosensory-evoked potentials. At the end of the study, histological images suggested spinal cord regeneration. Our present findings suggest the possible utility of perilesional intrathecal administration of autologous BMSC in patients with chronic paraplegia. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

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

    PubMed

    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.

  17. Assessing the potential of colony morphology for dissecting the CFU-F population from human bone marrow stromal cells.

    PubMed

    Gothard, D; Dawson, J I; Oreffo, R O C

    2013-05-01

    Mesenchymal stem cells (MSCs) provide an ideal cell source for bone tissue engineering strategies. However, bone marrow stromal cell (BMSC) populations that contain MSCs are highly heterogeneous expressing a wide variety of proliferative and differentiation potentials. Current MSC isolation methods employing magnetic-activated and fluorescent-activated cell sorting can be expensive and time consuming and, in the absence of specific MSC markers, fail to generate homogeneous populations. We have investigated the potential of various colony morphology descriptors to provide correlations with cell growth potential. Density-independent colony forming unit-fibroblastic (CFU-F) capacity is a MSC prerequisite and resultant colonies display an array of shapes and sizes that might be representative of cell function. Parent colonies were initially categorised according to their diameter and cell density and grouped before passage for the subsequent assessment of progeny colonies. Whereas significant morphological differences between distinct parent populations indicated a correlation with immunophenotype, enhanced CFU-F capacity was not observed when individual colonies were isolated according to these morphological parameters. Colony circularity, an alternative morphological measure, displayed a strong correlation with subsequent cell growth potential. The current study indicates the potential of morphological descriptors for predicting cell growth rate and suggests new directions for research into dissection of human BMSC CFU-F populations.

  18. Osteogenic Differentiation of Human and Ovine Bone Marrow Stromal Cells in response to β-Glycerophosphate and Monosodium Phosphate.

    PubMed

    Bottagisio, Marta; Lovati, Arianna B; Lopa, Silvia; Moretti, Matteo

    2015-08-01

    Bone defects are severe burdens in clinics, and thus cell therapy offers an alternative strategy exploiting the features of bone marrow stromal cells (BMSCs). Sheep are a suitable orthopedic preclinical model for similarities with humans. This study compares the influence of two phosphate sources combined with bone morphogenetic protein-2 (BMP-2) on the osteogenic potential of human and ovine BMSCs. β-Glycerophosphate (β-GlyP) and monosodium phosphate (NaH2PO4) were used as organic and inorganic phosphate sources. Osteogenic differentiation of the BMSCs was assessed by calcified matrix, alkaline phosphatase (ALP) activity, and gene expression analysis. A higher calcified matrix deposition was detected in BMSCs cultured with NaH2PO4. Although no significant differences were detected among media for human BMSCs, β-GlyP with or without BMP-2 determined a positive trend in ALP levels compared to NaH2PO4. In contrast, NaH2PO4 had a positive effect on ALP levels in ovine BMSCs. β-GlyP better supported the expression of COL1A1 in human BMSCs, whereas all media enhanced RUNX2 and SPARC expression. Ovine BMSCs responded poorly to any media for RUNX2, COL1A1, and SPARC expression. NaH2PO4 improved calcified matrix deposition without upregulating the transcriptional expression of osteogenic markers. A further optimization of differentiation protocols needs to be performed to translate the procedures from preclinical to clinical models.

  19. Characterization of the interface of the bone marrow stromal cell antigen 2-Vpu protein complex via computational chemistry.

    PubMed

    Zhou, Jinming; Zhang, Zhixin; Mi, Zeyun; Wang, Xin; Zhang, Quan; Li, Xiaoyu; Liang, Chen; Cen, Shan

    2012-02-14

    Bone marrow stromal cell antigen 2 (BST-2) inhibits the release of enveloped viruses from the cell surface. Various viral counter measures have been discovered, which allow viruses to escape BST-2 restriction. Human immunodeficiency virus type 1 (HIV-1) encodes viral protein U (Vpu) that interacts with BST-2 through their transmembrane domains and causes the downregulation of cell surface BST-2. In this study, we used a computer modeling method to establish a molecular model to investigate the binding interface of the transmembrane domains of BST-2 and Vpu. The model predicts that the interface is composed of Vpu residues I6, A10, A14, A18, V25, and W22 and BST-2 residues L23, I26, V30, I34, V35, L41, I42, and T45. Introduction of mutations that have been previously reported to disrupt the Vpu-BST-2 interaction led to a calculated higher binding free energy (MMGBSA), which supports our molecular model. A pharmacophore was also generated on the basis of this model. Our results provide a precise model that predicts the detailed interaction occurring between the transmembrane domains of Vpu and BST-2 and should facilitate the design of anti-HIV agents that are able to disrupt this interaction.

  20. Ternary composite scaffolds with tailorable degradation rate and highly improved colonization by human bone marrow stromal cells.

    PubMed

    Idaszek, J; Bruinink, A; Święszkowski, W

    2015-07-01

    Poly(ε-caprolactone), PCL, is of great interest for fabrication of biodegradable scaffolds due to its high compatibility with various manufacturing techniques, especially Fused Deposition Modeling (FDM). However, slow degradation and low strength make application of PCL limited only to longer-term bioresorbable and non-load bearing implants. To overcome latter drawbacks, ternary PCL-based composite fibrous scaffolds consisting of 70-95 wt % PCL, 5 wt % Tricalcium Phosphate (TCP) and 0-25 wt % poly(lactide-co-glycolide) (PLGA) were fabricated using FDM. In the present study, the effect of composition of the scaffolds on their mechanical properties, degradation kinetics, and surface properties (wettability, surface energy, and roughness) was investigated and correlated with response of human bone marrow mesenchymal stromal cells (HBMC). The presence of PLGA increased degradation kinetics, surface roughness and significantly improved scaffold colonization. Of the evaluated surface properties only the wettability was correlated with the surface area colonized by HBMC. This study demonstrates that introduction of PLGA into PCL-TCP binary composite could largely abolish the disadvantages of the PCL matrix and improve biocompatibility by increasing wettability and polar interactions rather than surface roughness. Additionally, we showed great potential of multicellular spheroids as a sensitive in vitro tool for detection of differences in chemistry of 3D scaffolds. © 2014 Wiley Periodicals, Inc.

  1. In vivo bone formation by human marrow stromal cells in biodegradable scaffolds that release dexamethasone and ascorbate-2-phosphate.

    PubMed

    Kim, Hyongbum; Suh, Hwal; Jo, Sangmee Ahn; Kim, Hyun Woo; Lee, Jung Min; Kim, Eun Hae; Reinwald, Yvonne; Park, Sang-Hyug; Min, Byoung-Hyun; Jo, Inho

    2005-07-15

    An unsolved problem with stem cell-based engineering of bone tissue is how to provide a microenvironment that promotes the osteogenic differentiation of multipotent stem cells. Previously, we fabricated porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds that released biologically active dexamethasone (Dex) and ascorbate-2-phosphate (AsP), and that acted as osteogenic scaffolds. To determine whether these osteogenic scaffolds can be used for bone formation in vivo, we seeded multipotent human marrow stromal cells (hMSCs) onto the scaffolds and implanted them subcutaneously into athymic mice. Higher alkaline phosphatase expression was observed in hMSCs in the osteogenic scaffolds compared with that of hMSCs in control scaffolds. Furthermore, there was more calcium deposition and stronger von Kossa staining in the osteogenic scaffolds, which suggested that there was enhanced mineralized bone formation. We failed to detect cartilage in the osteogenic scaffolds (negative Safranin O staining), which implied that there was intramembranous ossification. This is the first study to demonstrate the successful formation of mineralized bone tissue in vivo by hMSCs in PLGA scaffolds that release Dex and AsP.

  2. Glucocorticoid receptor Antagonist and siRNA Prevent Senescence of Human Bone Marrow Mesenchymal Stromal Cells in vitro

    PubMed Central

    Wei, Na; Yu, Yang; Joshi, Vijaya; Schmidt, Thomas; Qian, Fang; Salem, Aliasger K.; Stanford, Clark; Hong, Liu

    2016-01-01

    We investigated the effects mediated by glucocorticoid (GC) receptor (GR) blockage using RU486, a GR antagonist, and GR siRNA on the proliferative and differentiation capabilities of human bone marrow mesenchymal stromal/stem cells (MSCs), as well as on their senescence and antioxidant levels during extended in vitro culture. Treatment with either RU486 or GR siRNA for a 7 day period significantly increased the proliferation of MSCs as well as their osteogenic capabilities, as reflected by an increase in alkaline phosphatase (ALP) level after differentiation. After 4 weeks of the treatments MSCs improved or maintained their proliferation rates, while the control MSCs exhibited decreased proliferation. While all MSCs exhibited reduced osteogenic potential after 4 weeks of in vitro culture, the MSCs treated with GR inhibitors showed higher ALP levels than untreated MSCs after they were subjected to osteogenic differentiation. These treatment also significantly down-regulated the adipogenic capabilities of MSCs. Telomere lengths as well as the telomerase and superoxide dismutase activities of MSCs treated with either RU486 or GR siRNA appeared to be higher than those detected in controls. These results demonstrate that blockage of the effects mediated by the GCs normally found in fetal bovine serum may postpone senescence of these cells by up-regulating their antioxidant levels. These data suggested that blocking the effects mediated by GCs could potentially extend the lifespan of endogenous MSCs in patients who have elevated GC levels as a consequence of advancing age or estrogen depletion. PMID:23963647

  3. The interaction between bone marrow stromal cells and RGD-modified three-dimensional porous polycaprolactone scaffolds.

    PubMed

    Zhang, Huina; Lin, Chia-Ying; Hollister, Scott J

    2009-09-01

    We previously established a simple method to immobilize the Arg-Gly-Asp (RGD) peptide on polycaprolactone (PCL) two-dimensional film surfaces that significantly improved bone marrow stromal cell (BMSC) adhesion to these films. The current work extends this modification strategy to three-dimensional (3D) PCL scaffolds to investigate BMSC attachment, cellular distribution and cellularity, signal transduction and survival on the modified PCL scaffold compared to those on the untreated ones. The results demonstrated that treatment of 3D PCL scaffold surfaces with 1,6-hexanediamine introduced the amino functional groups onto the porous PCL scaffold homogenously as detected by a ninhydrin staining method. Followed by the cross-linking reaction, RGDC peptide was successfully immobilized on the surface of PCL scaffold. Although the static seeding method used in this study caused heterogeneous cell distribution, the RGD-modified PCL scaffold still demonstrated the improved BMSC attachment and cellular distribution in the scaffold. More importantly, the integrin-mediated signal transduction FAK-PI3K-Akt pathway was significantly up-regulated by RGD modification and a subsequent increase in cell survival and growth was found in the modified scaffold. The present study introduces an easy method to immobilize RGD peptide on the 3D porous PCL scaffold and provides further evidence that modification of 3D PCL scaffolds with RGD peptides elicits specific cellular responses and improves the final cell-biomaterial interaction.

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

    PubMed

    He, Xiaomin; Feng, Bei; Huang, Chuanpei; Wang, Hao; Ge, Yang; Hu, Renjie; Yin, Meng; Xu, Zhiwei; Wang, Wei; Fu, Wei; Zheng, Jinghao

    2015-01-01

    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.

  5. Establishment of a novel clonal murine bone marrow stromal cell line for assessment of p53 responses to genotoxic stress

    SciTech Connect

    Gorbunov, Nikolai V.; Morris, James E.; Greenberger, J S.; Thrall, Brian D.

    2002-10-15

    The p53 protein is widely regarded as an important sensor of genotoxic damage in cells, and mutations in p53 are the most frequent observed in human cancers. Rapid assays for evaluating the potential of a chemical or physical agent to alter the transcriptional regulatory role of p53 may therefore serve as useful tools in toxicological research. In this study, the use of enhanced green fluorescent protein (EGFP) as a live cell reporter to assess the transactivation response of p53 to chemical and physical agents was evaluated. A stable murine bone marrow stromal cell line (D2XRIIGFP24) expressing EGFP under control of p53 response elements was established. D2XRIIGFP24 cells displayed low constitutive background fluorescence which was significantly enhanced in response to exposure to agents that induced of p53 protein levels. Increases in EGFP fluorescence in response to oxidative and nitrosative stress as well as UVC irradiation were dose-dependent, detectable within 3 hours of expo sure and correlated closely with the amount of p53 protein accumulated within the cell. The results demonstrate the potential for rapid and early detection of p53 transactivation using the EGFP reporter approach and indicate this approach is adaptable to a variety of fluorescent assay techniques and a useful cell model for molecular toxicology research.

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

    PubMed Central

    Li, Hongzhe; Ghazanfari, Roshanak; Zacharaki, Dimitra; Ditzel, Nicholas; Isern, Joan; Ekblom, Marja; Méndez-Ferrer, Simón; Kassem, Moustapha; Scheding, Stefan

    2014-01-01

    Summary 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. PMID:25454633

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

    PubMed

    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.

  8. Repair of osteochondral defects with in vitro engineered cartilage based on autologous bone marrow stromal cells in a swine model

    PubMed Central

    He, Aijuan; Liu, Lina; Luo, Xusong; Liu, Yu; Liu, Yi; Liu, Fangjun; Wang, Xiaoyun; Zhang, Zhiyong; Zhang, Wenjie; Liu, Wei; Cao, Yilin; Zhou, Guangdong

    2017-01-01

    Functional reconstruction of large osteochondral defects is always a major challenge in articular surgery. Some studies have reported the feasibility of repairing articular osteochondral defects using bone marrow stromal cells (BMSCs) and biodegradable scaffolds. However, no significant breakthroughs have been achieved in clinical translation due to the instability of in vivo cartilage regeneration based on direct cell-scaffold construct implantation. To overcome the disadvantages of direct cell-scaffold construct implantation, the current study proposed an in vitro cartilage regeneration strategy, providing relatively mature cartilage-like tissue with superior mechanical properties. Our strategy involved in vitro cartilage engineering, repair of osteochondral defects, and evaluation of in vivo repair efficacy. The results demonstrated that BMSC engineered cartilage in vitro (BEC-vitro) presented a time-depended maturation process. The implantation of BEC-vitro alone could successfully realize tissue-specific repair of osteochondral defects with both cartilage and subchondral bone. Furthermore, the maturity level of BEC-vitro had significant influence on the repaired results. These results indicated that in vitro cartilage regeneration using BMSCs is a promising strategy for functional reconstruction of osteochondral defect, thus promoting the clinical translation of cartilage regeneration techniques incorporating BMSCs. PMID:28084417

  9. Limited Functional Effects of Subacute Syngeneic Bone Marrow Stromal Cell Transplantation After Rat Spinal Cord Contusion Injury.

    PubMed

    Sandner, Beatrice; Ciatipis, Mareva; Motsch, Melanie; Soljanik, Irina; Weidner, Norbert; Blesch, Armin

    2016-01-01

    Cell transplantation might be one means to improve motor, sensory, or autonomic recovery after traumatic spinal cord injury (SCI). Among the different cell types evaluated to date, bone marrow stromal cells (BMSCs) have received considerable interest due to their potential neuroprotective properties. However, uncertainty exists whether the efficacy of BMSCs after intraspinal transplantation justifies an invasive procedure. In the present study, we analyzed the effect of syngeneic BMSC transplantation following a moderate to severe rat spinal cord injury. Adult Fischer 344 rats underwent a T9 contusion injury (200 kDy) followed by grafting of GFP-expressing BMSCs 3 days postinjury. Animals receiving a contusion injury without cellular grafts or an injury followed by grafts of syngeneic GFP-expressing fibroblasts served as control. Eight weeks posttransplantation, BMSC-grafted animals showed only a minor effect in one measure of sensorimotor recovery, no significant differences in tissue sparing, and no changes in the recovery of bladder function compared to both control groups in urodynamic measurements. Both cell types survived in the lesion site with fibroblasts displaying a larger graft volume. Thus, contrary to some reports using allogeneic or xenogeneic transplants, subacute intraparenchymal grafting of syngeneic BMSCs has only a minor effect on functional recovery.

  10. The crucial role of vitamin C and its transporter (SVCT2) in bone marrow stromal cell autophagy and apoptosis

    PubMed Central

    Sangani, Rajnikumar; Periyasamy-Thandavan, Sudharsan; Pathania, Rajneesh; Ahmad, Saif; Kutiyanawalla, Ammar; Kolhe, Ravindra; Bhattacharyya, Maryka H.; Chutkan, Norman; Hunter, Monte; Hill, William D.; Hamrick, Mark; Isales, Carlos; Fulzele, Sadanand

    2016-01-01

    Vitamin C is an antioxidant that plays a vital role in various biological processes including bone formation. Previously, we reported that vitamin C is transported into bone marrow stromal cells (BMSCs) through the sodium dependent Vitamin C Transporter 2 (SVCT2) and this transporter plays an important role in osteogenic differentiation. Furthermore, this transporter is regulated by oxidative stress. To date, however, the exact role of vitamin C and its transporter (SVCT2) in ROS regulated autophagy and apoptosis in BMSCs is poorly understood. In the present study, we observed that oxidative stress decreased survival of BMSCs in a dose-dependent manner and induced growth arrest in the G1 phase of the cell cycle. These effects were accompanied by the induction of autophagy, confirmed by P62 and LC3B protein level and punctate GFP–LC3B distribution. The supplementation of vitamin C significantly rescued the BMSCs from oxidative stress by regulating autophagy. Knockdown of the SVCT2 transporter in BMSCs synergistically decreased cell survival even under low oxidative stress conditions. Also, supplementing vitamin C failed to rescue cells from stress. Our results reveal that the SVCT2 transporter plays a vital role in the mechanism of BMSC survival under stress conditions. Altogether, this study has given new insight into the role of the SVCT2 transporter in oxidative stress related autophagy and apoptosis in BMSCs. PMID:26210298

  11. The New Role of CD163 in the Differentiation of Bone Marrow Stromal Cells into Vascular Endothelial-Like Cells

    PubMed Central

    Lu, Wei; Su, Le; Yu, Zhezheng; Zhang, Shangli; Miao, Junying

    2016-01-01

    Bone marrow stromal cells (BMSCs) can differentiate into vascular endothelial cells (VECs). It is regarded as an important solution to cure many diseases, such as ischemic diseases and diabetes. However, the mechanisms underlying BMSC differentiation into VECs are not well understood. Recent reports showed that CD163 expression was associated with angiogenesis. In this study, overexpression of CD163 in BMSCs elevated the protein level of the endothelial-associated markers CD31, Flk-1, eNOS, and VE-cadherin, significantly increased the proportion of Alexa Fluor 488-acetylated-LDL-positive VECs, and promoted angiogenesis on Matrigel. Furthermore, we demonstrated that CD163 acted downstream homeobox containing 1 (Hmbox1) and upstream fibroblast growth factor 2 (FGF-2). These data suggested that CD163 was involved in Hmbox1/CD163/FGF-2 signal pathway in BMSC differentiation into vascular endothelial-like cells. We found a new signal pathway and a novel target for further investigating the gene control of BMSC differentiation into a VEC lineage. PMID:26880943

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

    PubMed

    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.

  13. Fetal Bone Marrow-Derived Mesenchymal Stem/Stromal Cells Enhance Humanization and Bone Formation of BMP7 Loaded Scaffolds.

    PubMed

    Shafiee, Abbas; Baldwin, Jeremy G; Patel, Jatin; Holzapfel, Boris M; Fisk, Nicholas M; Khosrotehrani, Kiarash; Hutmacher, Dietmar W

    2017-09-01

    Tissue engineered constructs built with human cells capable of generating a bone-like organ within the mouse have attracted considerable interest over the past decade. Here, we aimed to compare the utility of human mesenchymal stem/stromal cells (MSC) isolated from fetal term placenta (fPL-MSC) and fetal first trimester bone marrow (fBM-MSC) in a polycaprolactone scaffold/BMP7-based model in nude mice. Furthermore, fPL-MSC were co-seeded with fetal placenta-derived endothelial colony forming cells (ECFC) to assess the impact of ECFC on fPL-MSC osteogenesis. X-ray radiography and micro computed tomography analyses showed enhanced bone formation in all BMP7 groups; however there was no difference after 2 months in bone formation between scaffolds seeded with fPL-MSC alone or combination of ECFC and fPL-MSC. Of interest, fBM-MSC showed the highest level of bone formation. Additionally, endochondral ossification contributed in generation of bone in fBM-MSC. Histological analysis showed the primary role of BMP in generation of cortical and trabecular bone, and the recruitment of hematopoietic cells to the scaffolds. Current in vivo engineered bone organs can potentially be used for drug screening or as models to study bone tissue development in combination with haematopoiesis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.