Single-cell RNA sequencing on human osteoblasts
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE147390 | GEO | 2021/03/01
REPOSITORIES: GEO
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Project description:Purpose: We have used microarrays to identify gene expression profiles that distinguish mouse OS cells from normal pre-osteoblast cells and mature osteoblast cells. Methods: Transcriptional profiles of three cell lines derived from tumors from Osx-Cre p53fl/fl Rbfl/fl (fibroblastic OS) mouse model, and from pre-osteoblast cells (Kusa4b10 mouse bone marrow stromal cell line) and osteoblast cells (derived by in vitro differentiation of the Kusab410 mouse bone marrow stromal cell line) were generated by microarray analysis, each in triplicate, using Affymetrix mouse Gene1.0ST arrays. Transcriptional profiles were analyzed in cell lines derived from tumors from a genetically engineered mouse model of human osteosarcoma (Osx-Cre p53fl/fl Rbfl/fl) and osteoblast cells derived from the Kusa4b10 mouse bone marrow stromal cell line, in the undifferentiated state (pre-osteoblasts) and differentiated state (osteoblasts).
2015-01-29 | E-GEOD-63939 | biostudies-arrayexpress
Project description:Background:; Osteoblast differentiation requires the coordinated stepwise expression of multiple genes. Histone deacetylase inhibitors (HDIs) accelerate the osteoblast differentiation process by blocking the activity of histone deacetylases (HDACs), which alter gene expression by modifying chromatin structure. We previously demonstrated that HDIs and HDAC3 shRNAs accelerate matrix mineralization and the expression of osteoblast maturation genes (e.g. alkaline phosphatase, osteocalcin). Identifying other genes that are differentially regulated by HDIs might identify new pathways that contribute to osteoblast differentiation. Results:; To identify other osteoblast genes that are altered early by HDIs, we incubated MC3T3-E1 preosteoblasts with HDIs (trichostatin A, MS-275, or valproic acid) for 18 hours in osteogenic conditions. The promotion of osteoblast differentiation by HDIs in this experiment was confirmed by osteogenic assays. Gene expression profiles relative to vehicle-treated cells were assessed by microarray analysis with Affymetrix GeneChip 430 2.0 arrays. The regulation of several genes by HDIs in MC3T3-E1 cells and primary osteoblasts was verified by quantitative real-time PCR. Nine genes were differentially regulated by at least two-fold after exposure to each of the three HDIs and six were verified by PCR in osteoblasts. Four of the verified genes (solute carrier family 9 isoform 3 regulator 1 (Slc9a3r1), sorbitol dehydrogenase 1, a kinase anchor protein, and glutathione S-transferase alpha 4) were induced. Two genes (proteasome subunit, beta type 10 and adaptor-related protein complex AP-4 sigma 1) were suppressed. We also identified eight growth factors and growth factor receptor genes that are significantly altered by each of the HDIs, including Frizzled related proteins 1 and 4, which modulate the Wnt signaling pathway. Conclusions: This study identifies osteoblast genes that are regulated early by HDIs and indicates pathways that might promote osteoblast maturation following HDI exposure. One gene whose upregulation following HDI treatment is consistent with this notion is Slc9a3r1. Also known as NHERF1, Slc9a3r1 is required for optimal bone density. Similarly, the regulation of Wnt receptor genes indicates that this crucial pathway in osteoblast development is also affected by HDIs. These data support the hypothesis that HDIs regulate the expression of genes that promote osteoblast differentiation and maturation. Experiment Overall Design: To identify other osteoblast genes that are altered early by HDIs, we incubated MC3T3-E1 preosteoblasts with HDIs (trichostatin A, MS-275, or valproic acid) or the vehicle control (DMSO) for 18 hours in osteogenic conditions. Gene expression profiles relative to vehicle-treated cells were assessed in triplicate (in some cases quadruplicate) samples by microarray analysis with Affymetrix GeneChip 430 2.0 arrays.
2008-06-16 | E-GEOD-9247 | biostudies-arrayexpress
Project description:Gene expression analysis of murine MC3T3-E1 cells induced to undergo synchronized osteoblastic differentiation in vitro. MC3T3-E1 cells were cultured in vitro for up to 28 days in the presence of 8-glycerol phosphate and ascorbic acid to induce osteoblastic differentiation. Total RNA was isolated after 2, 5, 10 and 28 days in culture to sample proliferating preosteoblasts (Day 2), growth arrested preosteoblasts (Day 5), differentiating osteoblasts (Day 10), and mature osteoblasts (Day 28). Timing of sample collection was based on direct measurements of cell number, alkaline phosphatase expression, type 1A collagen synthesis, and matrix mineralization in parallel cultures. Triplicate samples from each time point were hybridized to slide arrays printed with the Operon Mouse Oligo set, version 2.0.
2012-06-27 | E-GEOD-30393 | biostudies-arrayexpress
Project description:Purpose: Osteoblast cells mature from a mesenchymal stem cell pool to become cells capable of forming bone matrix and mineralizing this matrix. The goal of this study was to characterize temporal changes in the transcriptome across osteoblast maturation, starting with committed mesenchymal stem cell/ early pre-osteoblast stage through to mature osteoblasts capable of matrix mineralization. Methods: Enriched populations of pre-osteoblast like cells were obtained from neonatal calvaria from C57BL/6J mice expressing CFP under the control of the Col3.6 promoter. These cells were placed into culture for 4 days, removed from culture and subjected FACS sorting based on the presence/absence of CFP expression. Cells expressing CFP were returned to culture, subjected to an osteoblast differentiation cocktail and RNA was collected at 2, 4, 6, 8, 10, 12, 14, 16 and 18 days post differentiation. Methods II: mRNA profiles for each time point were generated by next generation RNA sequencing, using an Illumina HiSeq 2000. Three technical replicates per samples were sequenced. The alignments for abundance estimation of transcripts was conducted using Bowtie version 0.12.9, using the NCBIm37 reference genome. Expression level per gene was calculated using RSEM version 1.2.0 with the parameters of --fragment-length-mean 280 and --fragment-length-sd 50, and the expression level for each sample was normalized relative to the per sample upper quartile. Gene expression in calvarial osteoblasts from neonatal C57BL/6J-Col3.6 CFP mice at 9 time points post differentiation
2014-06-23 | E-GEOD-54461 | biostudies-arrayexpress
Project description:Long noncoding RNAs (lncRNAs) have emerged as integral regulators of physiology and disease, but specific roles of lncRNAs in bone disease remain largely unknown. Here, we show that lnc-ob1 regulates osteoblast activity and bone formation in mice by upregulating the osteogenic transcription factor Osterix. Expression of lnc-ob1 is enriched in osteoblasts and upregulated during osteoblastogenesis. We demonstrate that osteoblast-specific knock-in of lnc-ob1 enhances bone formation and increases bone mass. Pharmacological overexpression of lnc-ob1 specifically in osteoblasts confers resistance to ovariectomy-induced osteoporosis in mice. In humans, expression of the homologue, lnc-OB1, decreases with age in osteoblasts of patients with osteoporosis. Mechanistically, lnc-ob1 upregulates the expression of Osterix in mouse and human osteoblasts, probably via inhibition of H3K27me3 methylation. Our data indicate that lnc-OB1 regulates bone formation and might be a drug target for the treatment of osteoporosis.
2019-03-28 | GSE112318 | GEO
Project description:Osteoblasts are the only somatic cell type with bone-forming ability. While the cellular and molecular mechanisms underlying their differentiation and function have been identified, their dynamic control in vivo is unclear. By intravital multiphoton microscopy of live bone tissues of osteoblast fluorescent reporter mice, we visualized intact mature osteoblasts in vivo and found that they actively secrete and capture extracellular microvesicles. Microvesicles from mature osteoblasts contain the microRNA miR-143-3p, which inhibits osteoblast differentiation. Osteoblast-specific deletion of miR-143 increased bone formation and miR-143-deficient microvesicles induced recovery from bone defect. In sum, we report a novel mode of intercellular communication in vivo via microRNA-containing extracellular vesicles, which controls bone homeostasis.
2022-02-04 | GSE144512 | GEO
Project description:Murine Runx3 is expressed in developing bone osteoblasts (OBLs) and its deletion in these cells culminates in severe congenital osteopenia. We demonstrate that Runx3 is non-redundantly involved in the proliferation of early pre-committed OBL progenitor cells, a critical step in the generation of adequate numbers of bone-forming OBLs. Thus, in the absence of Runx3 in cells of this lineage, the number of mature/active OBLs is significantly diminished, providing a mechanistic explanation to the observed osteopenia. This experiment was aimed at identifying genes that are likely regulated by Runx3 along osteoblastogenesis.
2014-05-01 | GSE57195 | GEO
Project description:Regeneration depends on the ability of mature cells at the injury site to respond to injury, generating tissue-specific progenitors that incorporate the blastema and proliferate to reconstitute the original organ architecture. The metabolic microenvironment has been tightly connected to cell function and identity during development and tumorigenesis. Yet, the link between metabolism and cell identity at the mechanistic level in a regenerative context remains unclear. The adult zebrafish caudal fin, and bone cells specifically, have been crucial for the understanding of mature cell contribution to tissue regeneration. Here, we use this model to explore the relevance of glucose metabolism for the cell fate transitions preceding new osteoblast formation and blastema assembly. We show that injury triggers a shift in the metabolic profile at early stages of regeneration, enhancing glycolysis at the expense of mitochondrial oxidation. This metabolic switch mediates transcriptional changes that make mature osteoblast amenable to be reprogramed into pre-osteoblasts and induces cell cycle re-entry and progression. Manipulation of the metabolic profile led to severe reduction of the pre-osteoblast pool, diminishing their capacity to generate new osteoblasts, and to a complete abrogation of blastema formation. Overall, our data indicate that metabolic alterations have a powerful instructive role in regulating genetic programs that dictate fate decisions and stimulate proliferation, thereby providing a deeper understanding on the mechanisms regulating blastema formation and bone regeneration.
2022-08-11 | GSE194385 | GEO
Project description:Osteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcription program essential for bone formation through both genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed. By performing genome-wide characterization of Runx2 binding at the three major stages of osteoblast differentiation: proliferation, matrix deposition and mineralization, we identified Runx2-dependent regulatory networks driving bone formation. Using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) over the course of these stages, we discovered close to 80,000 significantly enriched regions of Runx2 binding throughout the mouse genome. These binding events exhibited distinct patterns during osteogenesis, and were associated with proximal promoters as well as a large percentage of Runx2 occupancy in non-promoter regions: upstream, introns, exons, transcription termination site (TTS) regions, and intergenic regions. These peaks were partitioned into clusters that are associated with genes in complex biological processes that support bone formation. Using Affymetrix expression profiling of differentiating osteoblasts depleted of Runx2, we identified novel Runx2 targets including Ezh2, a critical epigenetic regulator; Crabp2, a retinoic acid signaling component; Adamts4 and Tnfrsf19, two remodelers of extracellular matrix. We demonstrated by luciferase assays that these novel biological targets are regulated by Runx2 occupancy at non-promoter regions. Our data establish that Runx2 interactions with chromatin across the genome reveal novel genes, pathways and transcriptional mechanisms that contribute to the regulation of osteoblastogenesis. MC3T3-E1 cells were treated with scramble or Runx2 shRNA, then harvested at proliferating stage (day 0) and differentiating stage (day 9). Total RNAs recovered from these cells were hybridization on Affymetrix microarrays. We sought to find new target genes or pathways regulated by Runx2 during osteoblast differentiation. When combined with genome-wide occupancy of Runx2, we expect to gain new insights on how Runx2 controls a transcriptional program essential for osteoblast differentiation.
2014-02-13 | E-GEOD-53982 | biostudies-arrayexpress
Project description:Sp7/Osterix is a master regulator of osteoblast specification. To identify the Sp7-mediated gene regulatory network in osteoblasts, we performed Sp7 ChIP-seq on primary mouse calvarial osteoblasts comparing the DNA binding profile with the transcriptional profile of Sp7-positive osteoblasts. Analysis of these identified a network of Sp7 regulated osteoblast targets and provides a new insight into the mode of Sp7 action in osteoblast. To further study for the Sp7 mode, we performed ChIP-seq for Sp1, Sp7, Dlx5, as a potential Sp7 partner identified in this study and mutated Sp7 which has mutations in the zinc finger domain, by using in vitro system with a pre-osteoblast mouse cell line, MC3T3E1.
2016-04-28 | GSE76185 | GEO