Generation and culture of osteoclasts.
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ABSTRACT:
SUBMITTER: Marino S
PROVIDER: S-EPMC4162467 | biostudies-other | 2014
REPOSITORIES: biostudies-other
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Project description:BACKGROUND:Infantile malignant osteopetrosis (IMO) is an autosomal recessive disorder characterized by non-functional osteoclasts and a fatal outcome early in childhood. About 50% of patients have mutations in the TCIRG1 gene. METHODS:IMO iPSCs were generated from a patient carrying a homozygous c.11279G>A (IVS18+1) mutation in TCIRG1 and transduced with a lentiviral vector expressing human TCIRG1. Embryoid bodies were generated and differentiated into monocytes. Non-adherent cells were harvested and further differentiated into osteoclasts on bovine bone slices. RESULTS:Release of the bone resorption biomarker CTX-I into the media of gene-corrected osteoclasts was 5-fold higher than that of the uncorrected osteoclasts and 35% of that of control osteoclasts. Bone resorption potential was confirmed by the presence of pits on the bones cultured with gene-corrected osteoclasts, absent in the uncorrected IMO osteoclasts. CONCLUSIONS:The disease phenotype was partially corrected in vitro, providing a valuable resource for therapy development for this form of severe osteopetrosis.
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Project description:Bone tissue undergoes constant remodeling and healing when fracture happens, in order to ensure its structural integrity. In order to better understand open biological and clinical questions linked to various bone diseases, bone cell co-culture technology is believed to shed some light into the dark. Osteoblasts/osteocytes and osteoclasts dominate the metabolism of bone by a multitude of connections. Therefore, it is widely accepted that a constant improvement of co-culture models with both cell types cultured on a 3D scaffold, is aimed to mimic an in vivo environment as closely as possible. Although in recent years a considerable knowledge of bone co-culture models has been accumulated, there are still many open questions. We here try to summarize the actual knowledge and address open questions.
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Project description:Autosomal recessive osteopetrosis is a human bone disease mainly caused by TCIRG1 gene mutations that prevent osteoclasts resorbing activity, recapitulated by the oc/oc mouse model. Bone marrow transplantation is the only available treatment, limited by the need for a matched donor. The use of induced pluripotent stem cells (iPSCs) as an unlimited source of autologous cells to generate gene corrected osteoclasts might represent a powerful alternative. We generated iPSCs from oc/oc mice, corrected the mutation using a BAC carrying the entire Tcirg1 gene locus as a template for homologous recombination, and induced hematopoietic differentiation. Similarly to physiologic fetal hematopoiesis, iPSC-derived CD41(+) cells gradually gave rise to CD45(+) cells, which comprised both mature myeloid cells and high proliferative potential colony-forming cells. Finally, we differentiated the gene corrected iPSC-derived myeloid cells into osteoclasts with rescued bone resorbing activity. These results are promising for a future translation into the human clinical setting.
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Project description:BackgroundCultivation of osteoclasts is a basic tool for investigating osteolytic bone diseases. Fetal bovine serum (FBS) is the standard supplement used for in vitro cell culture medium. Typically, the serum volume fraction used for osteoclast cultivation is 10%. In this study, we investigated the use of a low serum (1% FBS) model for culturing osteoclasts.ResultsTo confirm the validity of this model for use in osteoclast research, we compared the capacity for osteoclastogenesis and bone resorption of RANKL-induced RAW 264.7 cells cultured in medium supplemented with 10% FBS and 1% FBS. Osteoclasts were successfully generated in medium supplemented with 1% FBS, and exhibited prolonged longevity and similar bone resorbing ability to those generated in medium supplemented with 10% FBS, although the osteoclasts were smaller in size. Proteomics and bioinformatics analyses were performed to assess the suitability of osteoclasts formed in low serum-containing medium for use in research focusing on osteoclast differentiation and function. Our study demonstrated that a total of 100 proteins were differentially expressed in cells cultured in medium containing 1% FBS, of which 29 proteins were upregulated, and 71 proteins were downregulated. Bioinformatics analysis showed that the electron transport chain and oxidative phosphorylation pathways were downregulated obviously; however, the osteoclast signaling pathway was unaffected. The data have been deposited to the ProteomeXchange with identifier PXD001935.ConclusionOur study provides clear evidence of the validity of the low serum model for use in studying RANKL-dependent osteoclasts differentiation and bone resorption with the advantage of prolonged survival time.
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Project description:BackgroundThe present study was designed to examine the role of isoflavone genistein (GS) on bone formation, regulating oxidative stress and cell cycle in primary osteoblasts, as well as attenuation of osteoclast formation.MethodsPrimary calvaria osteoblasts were isolated from 2 to 3?days old neonatal rat pups (n?=?6-8) of Sprague Dawley rats. Osteoblasts were incubated with varying concentrations of GS and different assays viz. cell proliferation, differentiation, calcium deposition, cell cycle progression, antioxidant ability, and osteogenic gene expression were performed. Tartrate-resistant acid phosphatase (TRAP) staining and immunolocalization of cathepsin K protein were assessed in bone marrow-derived osteoclasts.ResultsResults revealed that GS markedly induced cell growth and osteoblast differentiation depending upon dose. The fluorescent dye DCFH-DA staining data proved the antioxidant ability of GS, which reduced the H2O2- induced intracellular oxidative stress in osteoblasts. Quantitative real-time PCR analysis revealed that GS treatment upregulated the expression of osteoblastic genes of Runt-related transcription factor 2 (Runx2), bone morphogenetic proteins 2 (BMP2), and osteocalcin. Immunolocalization of BMP2 also indicated the osteogenic efficacy of GS. Furthermore, TRAP staining and cathepsin K expression depicted that GS inhibited multinucleated osteoclasts formation.ConclusionsIn conclusion, GS isoflavone might impart protective effects against oxidative stress-induced bone loss and thus, could maintain skeletal growth.
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Project description:Clinically, osteolytic phenotype is rare in prostate cancer. The molecular mechanism of bone metastasis in PCa is not fully understood. We performed RNA-seq to identify osteogenic and tumor associated roles in prostate cancer by a co-culture of osteoblasts (MG63) and prostate cancer cells (C4-2, C4-2B, 22Rv1 and DU145). We compared osteoblastic prostate cancer with osteolytic prostate cancer to evaluate the difference in phenotype of bone metastasis.
2023-09-26 | GSE210729 | GEO
Project description:Astronauts are at risk of losing 1.0% to 1.5% of their bone mass for every month they spend in space despite their adherence to diets and exercise regimens designed to protect their musculoskeletal systems. This loss is the result of microgravity-related impairment of osteocyte and osteoblast function and the consequent upregulation of osteoclast-mediated bone resorption. This review describes the ontogeny of osteoclast hematopoietic stem cells and the contributions macrophage colony stimulating factor, receptor activator of the nuclear factor-kappa B ligand, and the calcineurin pathways make in osteoclast differentiation and provides details of bone formation, the osteoclast cytoskeleton, the immune regulation of osteoclasts, and osteoclast mechanotransduction on Earth, in space, and under conditions of simulated microgravity. The article discusses the need to better understand how osteoclasts are able to function in zero gravity and reviews current and prospective therapies that may be used to treat osteoclast-mediated bone disease.
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Project description:To understand how interactions of myeloma cells with osteoclasts and mesenchymal stem cells in the bone marrow affect the clinical course of myeloma, we used microarrays to study changes in gene expression in freshly isolated myeloma plasma cells following co-cultures with osteoclasts (8 experiments) or with mesenchymal stem cells (13 experiments). Interaction with osteoclasts induced changes in the expression of 675 genes, and interaction with mesenchymal stem cells induced changes in the expression of 296 genes. Expression of only 58 genes commonly and similarly changed in both co-culture systems. Among these, we identified genes associated with overall, progression-free, and post-relapse survival, and developed survival prediction models. Gene expression data from 347 patients treated with total therapy 2 protocol, 433 with total therapy 3, and 98 patients who received various treatments (91 of them high-dose therapy with autologous stem cell support) were used for the analysis. Good predictive models were developed only for post-relapse survival, using genes involved in interaction with osteoclasts or with mesenchymal stem cells. The best predictive model used expression of first relapse of 33 probesets whose expression changed in myeloma cells following interaction with osteoclasts, with hazard ratios of 24, 20, and 12 for patients who relapsed following total therapy 2, total therapy 3 and the various other treatments, respectively. Among the probesets used for prediction, only 10, representing 8 genes, were commonly changed after both co-culture systems. These could present favorable target for therapy. Global gene expression profiling of osteoclasts (OCs) before and after co-culture with primary multiple myeloma plasma cells (MMPCs) was done using Affymetrix microarrays. Eight MMPC and OC co-culture experiments were performed using MMPC isolated from 8 patients and OC prepared from 8 different patients.
2015-08-13 | E-GEOD-31156 | biostudies-arrayexpress
Project description:To understand how interactions of myeloma cells with osteoclasts and mesenchymal stem cells in the bone marrow affect the clinical course of myeloma, we used microarrays to study changes in gene expression in freshly isolated myeloma plasma cells following co-cultures with osteoclasts (8 experiments) or with mesenchymal stem cells (13 experiments). Interaction with osteoclasts induced changes in the expression of 675 genes, and interaction with mesenchymal stem cells induced changes in the expression of 296 genes. Expression of only 58 genes commonly and similarly changed in both co-culture systems. Among these, we identified genes associated with overall, progression-free, and post-relapse survival, and developed survival prediction models. Gene expression data from 347 patients treated with total therapy 2 protocol, 433 with total therapy 3, and 98 patients who received various treatments (91 of them high-dose therapy with autologous stem cell support) were used for the analysis. Good predictive models were developed only for post-relapse survival, using genes involved in interaction with osteoclasts or with mesenchymal stem cells. The best predictive model used expression of first relapse of 33 probesets whose expression changed in myeloma cells following interaction with osteoclasts, with hazard ratios of 24, 20, and 12 for patients who relapsed following total therapy 2, total therapy 3 and the various other treatments, respectively. Among the probesets used for prediction, only 10, representing 8 genes, were commonly changed after both co-culture systems. These could present favorable target for therapy. Global gene expression profiling of osteoclasts (OCs) before and after co-culture with primary multiple myeloma plasma cells (MMPCs) was done using Affymetrix microarrays.
2015-08-13 | GSE31156 | GEO
Project description:To understand how interactions of myeloma cells with osteoclasts and mesenchymal stem cells in the bone marrow affect the clinical course of myeloma, we used microarrays to study changes in gene expression in freshly isolated myeloma plasma cells following co-cultures with osteoclasts (8 experiments) or with mesenchymal stem cells (13 experiments). Interaction with osteoclasts induced changes in the expression of 675 genes, and interaction with mesenchymal stem cells induced changes in the expression of 296 genes. Expression of only 58 genes commonly and similarly changed in both co-culture systems. Among these, we identified genes associated with overall, progression-free, and post-relapse survival, and developed survival prediction models. Gene expression data from 347 patients treated with total therapy 2 protocol, 433 with total therapy 3, and 98 patients who received various treatments (91 of them high-dose therapy with autologous stem cell support) were used for the analysis. Good predictive models were developed only for post-relapse survival, using genes involved in interaction with osteoclasts or with mesenchymal stem cells. The best predictive model used expression of first relapse of 33 probesets whose expression changed in myeloma cells following interaction with osteoclasts, with hazard ratios of 24, 20, and 12 for patients who relapsed following total therapy 2, total therapy 3 and the various other treatments, respectively. Among the probesets used for prediction, only 10, representing 8 genes, were commonly changed after both co-culture systems. These could present favorable target for therapy. Global gene expression profiling of primary multiple myeloma plasma cells (MMPCs) before and after interaction with osteoclasts (OCs) was done using Affymetrix microarrays.
2019-08-21 | GSE31154 | GEO