Lineage-committed osteoclast precursors circulate in blood and settle down into bone
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ABSTRACT: Osteoclasts are derived from the monocyte/macrophage lineage, but little is known about osteoclast precursors in circulation. Bone marrow cells were subdivided into three populations; RANKhighFmslow, RANKhighFmshigh and RANKlowFmshigh. GeneChip analysis confirmed that the expression levels of monocyte-macrophage markers such as Emr1 (F4/80), Itgam (CD11b) and Csf1 (c-Fms) were lower in the RANKhighFmslow than RANKlowFmshigh population. In contrast, cells in the RANKhighFmslow population expressed higher levels of osteoclast markers such as Car ll (carbonic anhydrase ll), Mmp9 (matrix metalloproteinase 9), Acp5 (acid phosphatase 5) and Tfrc (transferrin receptor). These results suggest that RANKhighFmslow cells express few of the phenotypes of monocytes, and their differentiation into osteoclasts occurs at a slightly more advanced stage than that of the RANKlowFmshigh population.
Project description:Osteoclasts are derived from the monocyte/macrophage lineage, but little is known about osteoclast precursors in circulation. Bone marrow cells were subdivided into three populations; RANKhighFmslow, RANKhighFmshigh and RANKlowFmshigh. GeneChip analysis confirmed that the expression levels of monocyte-macrophage markers such as Emr1 (F4/80), Itgam (CD11b) and Csf1 (c-Fms) were lower in the RANKhighFmslow than RANKlowFmshigh population. In contrast, cells in the RANKhighFmslow population expressed higher levels of osteoclast markers such as Car ll (carbonic anhydrase ll), Mmp9 (matrix metalloproteinase 9), Acp5 (acid phosphatase 5) and Tfrc (transferrin receptor). These results suggest that RANKhighFmslow cells express few of the phenotypes of monocytes, and their differentiation into osteoclasts occurs at a slightly more advanced stage than that of the RANKlowFmshigh population. RANKhighFmslow cells and RANKlowFmshigh cells were isolated from bone marrow in ddY mice by FACS (Fluorescent activated cell sorting). Differential expression levels of mRNA were determined by GeneChip analysis.
Project description:Epigenetic regulation is a fundamental mechanism mediating various cellular processes. However, epigenetic mechanisms in osteoclastogenesis remain to be elucidated. We performed microarray analysis to investigate gene expression in osteoclasts derived from wild-type and Dnmt3aknockout mice. In vitro osteoclast culture were performed using wild-type control and Dnmt3a knockout bone marrow-derived monocyte/macrophage precursor cells.
Project description:Comparison of gene expression of the osteoclast precursor myeloid blast seeded on plastic and on bone, primed with M-CSF for 4 days and culture with M-CSF and RANKL for 1 day. Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone. Two conditions: Osteoclast precursors on plastic and on bone, n=4, dye swap
Project description:In response to the cytokines, macrophage colony-stimulating factor and receptor activator of NF-kB ligand, monocyte precursors differentiate into bone marrow-derived macrophages (BMDMs) that ultimately fuse to form multi-nucleated osteoclasts, following a tightly controlled genetic program where specific sets of genes are differentially expressed. We used microarrays to examine the gene expression profile underlying mouse osteoclast differentiation.
Project description:To survey the proteome of osteoclast secretory lysosomes, we used superparamagnetic iron oxide nanoparticles (SPIONs) to enrich for these endo-lysosomal-related organelles from murine osteoclast cultures. Briefly, large scale murine bone marrow monocyte (BMM)-derived osteoclast cultures were ‘pulsed’ with SPIONs to encourage uptake into endosomes and then ‘chased’ into secretory lysosomes upon the convergence of SPION-loaded endosomes with lysosomes and secretory pathways. Following the ‘pulse-chase’, osteoclasts were homogenized, SPION-loaded organelles captured-from post-nuclear supernatants using magnetic columns, and enriched organelles eluted and processed for 1D in-gel digestion and mass spectrometry.
Project description:To survey the proteome of osteoclast secretory lysosomes, we used superparamagnetic iron oxide nanoparticles (SPIONs) to enrich for these endo-lysosomal-related organelles from murine osteoclast cultures. Briefly, large scale murine bone marrow monocyte (BMM)-derived osteoclast cultures were ‘pulsed’ with SPIONs to encourage uptake into endosomes and then ‘chased’ into secretory lysosomes upon the convergence of SPION-loaded endosomes with lysosomes and secretory pathways. Following the ‘pulse-chase’, osteoclasts were homogenized, SPION-loaded organelles captured-from post-nuclear supernatants using magnetic columns, and enriched organelles eluted and processed for 1D in-gel digestion and mass spectrometry.
Project description:Colony Stimulating Factor 1(CSF1) is known to promote osteoclast progenitor survival but its role in regulating osteoclast differentiation and mature osteoclast function are less well understood. Macrophages have the potential to differentiate into osteoclasts and are also considered as osteoclast precursors. The microarray screen was designed to identify potential CSF1 targets in macrophage and osteoclast lineage.
Project description:To examine the miRNA changes that occur during osteoclast differentiation we used the miRCURY LNATM microRNA Array (7th Gen) to profile monocyte-derived osteoclasts and monocyte-derived macrophages. Of the 2046 probes present on the assay, we detected expression of ~400 (20%) miRNAs across all samples.
Project description:Comparison of gene expression of the osteoclast precursor myeloid blast seeded on plastic and on bone, primed with M-CSF for 4 days and culture with M-CSF and RANKL for 1 day. Osteoclasts and macrophages share progenitors that must receive decisive lineage signals driving them into their respective differentiation routes. Macrophage colony stimulation factor M-CSF is a common factor; bone is likely the stimulus for osteoclast differentiation. To elucidate the effect of both, shared mouse bone marrow precursor myeloid blast was pre-cultured with M-CSF on plastic and on bone. M-CSF priming prior to stimulation with M-CSF and osteoclast differentiation factor RANKL resulted in a complete loss of osteoclastogenic potential without bone. This coincided with a steeply decreased expression of osteoclast genes TRACP and DC-STAMP, but an increased expression of the macrophage markers F4/80 and CD11b. Compellingly, M-CSF priming on bone accelerated the osteoclastogenic potential: M-CSF primed cells that had received only one day M-CSF and RANKL and were grown on bone already expressed an array of genes that are associated with osteoclast differentiation and these cells differentiated into osteoclasts within 2 days. This implies that adhesion to bone dictates the fate of osteoclast precursors. Common macrophage-osteoclast precursors may become insensitive to differentiate into osteoclasts and regain osteoclastogenesis when bound to bone or when in the vicinity of bone.
Project description:Macrophages are a heterogeneous cell type implicated in injury, repair, and fibrosis after AKI, but the macrophage population associated with each phase is unclear.results of this study in a renal ischemia-reperfusion injury model allow phenotype and function to be assigned to CD11b+/Ly6C+ monocyte/macrophage populations in the pathophysiology of disease after AKI. we used a renal bilateral ischemia-reperfusion injury mouse model to identify unique monocyte/macrophage populations by differential expression of Ly6C in CD11b+Â cells and to define the function of these cells in the pathophysiology of disease on the basis of microarray gene signatures and reduction strategies Macrophage populations were sorted by Flow Cytometry into low and intermediate populations by Itgam(Cd11b) and Ly6c markers. The cells obtained in 5 weeks sham, 5 weeks IR, 9 day sham, and 9 day IR with 6 samples per group (3 int and 3 low). Cells were sorted in 350ul of RLT lysing buffer and kept at -80c until RNA extraction.Sample amplification, fragmentation, hybridization,washing and scanning were performed according to validated Affymetrix protocol in a CLIA certified lab.