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Human Bone Marrow Stromal Cells (human + bone_marrow_stromal_cell)
Selected AbstractsHuman tissue-engineered bone produced in clinically relevant amounts using a semi-automated perfusion bioreactor system: a preliminary studyJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 1 2010F. W. Janssen Abstract The aim of this study was to evaluate a semi-automated perfusion bioreactor system for the production of clinically relevant amounts of human tissue-engineered bone. Human bone marrow stromal cells (hBMSCs) of eight donors were dynamically seeded and proliferated in a perfusion bioreactor system in clinically relevant volumes (10 cm3) of macroporous biphasic calcium phosphate scaffolds (BCP particles, 2,6 mm). Cell load and distribution were shown using methylene blue staining. MTT staining was used to demonstrate viability of the present cells. After 20 days of cultivation, the particles were covered with a homogeneous layer of viable cells. Online oxygen measurements confirmed the proliferation of hBMSCs in the bioreactor. After 20 days of cultivation, the hybrid constructs became interconnected and a dense layer of extracellular matrix was present, as visualized by scanning electron microscopy (SEM). Furthermore, the hBMSCs showed differentiation towards the osteogenic lineage as was indicated by collagen type I production and alkaline phosphatase (ALP) expression. We observed no significant differences in osteogenic gene expression profiles between static and dynamic conditions like ALP, BMP2, Id1, Id2, Smad6, collagen type I, osteocalcin, osteonectin and S100A4. For the donors that showed bone formation, dynamically cultured hybrid constructs showed the same amount of bone as the statically cultured hybrid constructs. Based on these results, we conclude that a semi-automated perfusion bioreactor system is capable of producing clinically relevant and viable amounts of human tissue-engineered bone that exhibit bone-forming potential after implantation in nude mice. Copyright © 2009 John Wiley & Sons, Ltd. [source] Identifying a molecular phenotype for bone marrow stromal cells with in vivo bone-forming capacityJOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2010Kenneth H Larsen Abstract The ability of bone marrow stromal cells (BMSCs) to differentiate into osteoblasts is being exploited in cell-based therapy for repair of bone defects. However, the phenotype of ex vivo cultured BMSCs predicting their bone-forming capacity is not known. Thus we employed DNA microarrays comparing two human bone marrow stromal cell (hBMSC) populations: One is capable of in vivo heterotopic bone formation (hBMSC-TERT+Bone), and the other is not (hBMSC-TERT,Bone). Compared with hBMSC-TERT,Bone, the hBMSC-TERT+Bone cells had an increased overrepresentation of extracellular matrix genes (17% versus 5%) and a larger percentage of genes with predicted SP3 transcription factor,binding sites in their promoter region (21% versus 8%). On the other hand, hBMSC-TERT,Bone cells expressed a larger number of immune-response-related genes (26% versus 8%). In order to test for the predictive value of these markers, we studied the correlation between their expression levels in six different hBMSC-derived clones and the ability to form bone in vivo. We found a significant correlation for decorin, lysyl oxidase-like 4, natriuretic peptide receptor C, and tetranectin. No significant positive correlation was found for canonical osteoblastic markers Runx2, alkaline phosphatase, collagen type I, osteopontin, and bone sialoprotein. Prospective isolation of four additional hBMSC clones based on their expression levels of the molecular markers correlated with their in vivo bone-formation ability. In conclusion, our data suggest an in vitro molecular signature predictive for hBMSCs' in vivo bone-formation ability. Identifying more of these predictive markers would be very useful in the quality control of osteoblastic cells before use in therapy. © 2010 American Society for Bone and Mineral Research [source] Inhibition of Lamin A/C Attenuates Osteoblast Differentiation and Enhances RANKL-Dependent Osteoclastogenesis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2009Martina Rauner Abstract Age-related osteoporosis is characterized by low bone mass, poor bone quality, and impaired osteoblastogenesis. Recently, the Hutchinson-Gilford progeria syndrome (HGPS), a disease of accelerated aging and premature osteoporosis, has been linked to mutations in the gene encoding for the nuclear lamina protein lamin A/C. Here, we tested the hypothesis that inhibition of lamin A/C in osteoblastic lineage cells impairs osteoblastogenesis and accelerates osteoclastogenesis. Lamin A/C was knocked-down with small interfering (si)RNA molecules in human bone marrow stromal cells (BMSCs) differentiating toward osteoblasts. Lamin A/C knockdown led to an inhibition of osteoblast proliferation by 26% and impaired osteoblast differentiation by 48% based on the formation of mineralized matrix. In mature osteoblasts, expression levels of runx2 and osteocalcin mRNA were decreased by lamin A/C knockdown by 44% and 78%, respectively. Furthermore, protein analysis showed that osteoblasts with diminished levels of lamin A/C also secreted less osteocalcin and expressed a lower alkaline phosphatase activity (,50%). Lamin A/C inhibition increased RANKL mRNA and protein levels, whereas osteoprotegerin (OPG) expression was decreased, resulting in an increased RANKL/OPG ratio and an enhanced ability to support osteoclastogenesis, as reflected by a 34% increase of TRACP+ multinucleated cells. Our data indicate that lamin A/C is essential for proper osteoblastogenesis. Moreover, lack of lamin A/C favors an osteoclastogenic milieu and contributes to enhanced osteoclastogenesis. [source] Intracrine androgenic apparatus in human bone marrow stromal cellsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 9b 2009Tarvo Sillat Abstract It was suggested that human mesenchymal stromal cells might contain an intracrine enzyme machinery potentially able to synthesize the cell's own supply of dihydrotestosterone (DHT) from dehydroepiandrosterone (DHEA) pro-hormone produced in the adrenal cortex in the reticular zone, which is unique to primates. Indeed, 3,-hydroxysteroid dehydrogenase (3,-HSD) and 5,-reductase enzyme proteins were expressed in resting mesenchymal stromal cells (MSCs) in vitro. However, the ,bridging' enzymes 17,-HSDs, catalysing interconversion between 17,-ketosteroids and 17,-hydroxysteroids, were not found in resting MSCs, but 17,-HSD enzyme protein was induced in a dose-dependent manner by DHEA. Quantitative real-time polymerase chain reactions disclosed that this was mainly due to induction of the isoform 5 catalysing this reaction in ,forward', androgen-bound direction (P < 0.01). This work demonstrates that the MSCs have an intracrine machinery to convert DHEA to DHT if and when challenged by DHEA. DHEA as substrate exerts a positive, feed-forward up-regulation on the 17,-hydroxy steroid dehydrogenase-5, which may imply that DHEA-DHT tailor-making in MSCs is subjected to chronobiological regulation. [source] Adenovirus-mediated BMP2 expression in human bone marrow stromal cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2001Elizabeth A. Olmsted Abstract Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 ,M dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 ,g/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 ,M dexamethasone was reduced if the cells were not given 50 ,g/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells. J. Cell. Biochem. 82: 11,21, 2001. © 2001 Wiley-Liss, Inc. [source] Effects of peripheral benzodiazepine receptor ligands on proliferation and differentiation of human mesenchymal stem cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2004D.H. Lee The peripheral benzodiazepine receptor (PBR) has been known to have many functions such as a role in cell proliferation, cell differentiation, steroidogenesis, calcium flow, cellular respiration, cellular immunity, malignancy, and apoptosis. However, the presence of PBR has not been examined in mesenchymal stem cells. In this study, we demonstrated the expression of PBR in human bone marrow stromal cells (hBMSCs) and human adipose stromal cells (hATSCs) by RT-PCR and immunocytochemistry. To determine the roles of PBR in cellular functions of human mesenchymal stem cells (hMSCs), effects of diazepam, PK11195, and Ro5-4864 were examined. Adipose differentiation of hMSCs was decreased by high concentration of PBR ligands (50 ,M), whereas it was increased by low concentrations of PBR ligands (<10 ,M). PBR ligands showed a biphasic effect on glycerol-3-phosphate dehydrogenase (GPDH) activity. High concentration of PBR ligands (from 25 to 75 ,M) inhibited proliferation of hMSCs. However, clonazepam, which does not have an affinity to PBR, did not affect adipose differentiation and proliferation of hMSCs. The PBR ligands did not induce cell death in hMSCs. PK11195 (50 ,M) and Ro5-5864 (50 ,M) induced cell cycle arrest in the G2/M phase. These results indicate that PBR ligands play roles in adipose differentiation and proliferation of hMSCs. J. Cell. Physiol. 198: 91,99, 2004. © 2003 Wiley-Liss, Inc. [source] Comparative analysis of neuroectodermal differentiation capacity of human bone marrow stromal cells using various conversion protocolsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2006Andreas Hermann Abstract Human adult bone marrow-derived mesodermal stromal cells (hMSCs) are able to differentiate into multiple mesodermal tissues, including bone and cartilage. There is evidence that these cells are able to break germ layer commitment and differentiate into cells expressing neuroectodermal properties. There is still debate about whether this results from cell fusion, aberrant marker gene expression or real neuroectodermal differentiation. Here we extend our work on neuroectodermal conversion of adult hMSCs in vitro by evaluating various epigenetic conversion protocols using quantitative RT-PCR and immunocytochemistry. Undifferentiated hMSCs expressed high levels of fibronectin as well as several neuroectodermal genes commonly used to characterize neural cell types, such as nestin, ,-tubulin III, and GFAP, suggesting that hMSCs retain the ability to differentiate into neuroectodermal cell types. Protocols using a direct differentiation of hMSCs into a neural phenotype failed to induce significant changes in morphology and/or expression of markers of early and mature glial/neuronal cells types. In contrast, a multistep protocol with conversion of hMSCs into a neural stem cell-like population and subsequent terminal differentiation in mature glia and neurons generated relevant morphological changes as well as significant increase of expression levels of marker genes for early and late neural cell types, such as nestin, neurogenin2, MBP, and MAP2ab, accompanied by a loss of their mesenchymal properties. Our data provide an impetus for differentiating hMSCs in vitro into mature neuroectodermal cells. Neuroectodermally converted hMSCs may therefore ultimately help in treating acute and chronic neurodegenerative diseases. Analysis of marker gene expression for characterization of neural cells derived from MSCs has to take into account that several early and late neuroectodermal genes are already expressed in undifferentiated MSCs. © 2006 Wiley-Liss, Inc. [source] Manganese-guided cellular MRI of human embryonic stem cell and human bone marrow stromal cell viabilityMAGNETIC RESONANCE IN MEDICINE, Issue 4 2009Mayumi Yamada Abstract This study investigated the ability of MnCl2 as a cellular MRI contrast agent to determine the in vitro viability of human embryonic stem cells (hESC) and human bone marrow stromal cells (hBMSC). Basic MRI parameters including T1 and T2 values of MnCl2 -labeled hESC and hBMSC were measured and viability signal of manganese (Mn2+)-labeled cells was validated. Furthermore, the biological activity of Ca2+ -channels was modulated utilizing both Ca2+ -channel agonist and antagonist to evaluate concomitant signal changes. Metabolic effects of MnCl2 -labeling were also assessed using assays for cell viability, proliferation, and apoptosis. Finally, in vivo Mn2+ -guided MRI of the transplanted hESC was successfully achieved and validated by bioluminescence imaging. Magn Reson Med, 2009. © 2009 Wiley-Liss, Inc. 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