Project description:We report on explant osteoblast cultures from human patients, demonstrating that there are at least three sub-types of non-syndromic craniosynostosis defined by similarity of gene expression profiles.
Project description:We report on explant osteoblast cultures from human patients, demonstrating that there are at least three sub-types of non-syndromic craniosynostosis defined by similarity of gene expression profiles. Osteoblast growth in culture, 23 craniosynostosis skull samples (7 metopic; 8 coronal; 3 lambdoid; 5 sagittal) and 8 normal (4 cranial bones and 4 long bones)
Project description:Comparison of four craniosynostosis disease groups of 10 samples each: Apert syndrome (AS), Muenke syndrome (MS), Saetre-Chotzen syndrome (SCS), and non-syndromic craniosynostosis (NSS).
Project description:Assaying gene expression in sutural bone fragments from patients diagnosed with non-syndromic craniosynostosis. Sutural fragments were collected from both the fused and patent cranial suture of infants during cranial vault reconstruction. Gene expression was compared between the patent and fused sutures using the paired t-test. The aim was to identify thoses genes significantly differentially expressed in fused suture relative to patent.
Project description:Assaying gene expression in sutural bone fragments from patients diagnosed with non-syndromic craniosynostosis. Sutural fragments were collected from both the fused and patent cranial suture of infants during cranial vault reconstruction. Gene expression was compared between the patent and fused sutures using the paired t-test. The aim was to identify thoses genes significantly differentially expressed in fused suture relative to patent. Total RNA isolated from patent and fused human cranial sutures was assayed. Expression in synostosed suture was compared to patent suture.
Project description:Craniosynostosis (CS) is the congenital premature fusion of one or more cranial sutures and represents the more prevalent craniofacial malformation in humans, with an overall incidence of 1 out of 2000-3000 live births. Non-syndromic craniosynostoses (NSC) are believed to be multifactorial disorders, with a strong genetic component, due to possible gene–gene or gene–environment interactions that remain to be clearly identified. In this study we delved into the molecular signaling acting in calvarial tissue and cells from patients affected by nonsynodromic midline craniosynostosis, using a comparative analysis between fused and unfused sutures of each affected individuals. Using comparative microarray tissue gene expression profiling we have identified a subset of genes involved in the structure and function of the primary cilium, including the Bardet-Biedl syndrome 9 (BBS9) gene, which was recently associated to sagittal synostosis in a GWAS study. We therefore characterized BBS9 expression and cilium-related signaling in cells isolated from patients’ calvarial bone.
Project description:Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. In order to uncover the cellular diversity within sutures, we conducted single-cell transcriptomic and histological analyses of the embryonic murine coronal suture. We identify Erg and Pthlh as early markers of osteogenic progenitors in sutures, and distinct pre-osteoblast signatures between the bone fronts and periosteum. diverse mesenchymal layers at the coronal suture, including multiple distinct meningeal layers below the suture, and ligamentous, ectocranial, and hypodermal layers above the sutureIn the ectocranial layers above the suture, we observe a ligament-like population spanning the frontal and parietal bones and expressing genes implicated in mechanosensation. Mesenchyme in and around the coronal suture is asymmetrically distributed between the frontal and parietal bones, and we identify different states of osteogenic cells extending from the bone fronts into the more mature bone, and a potential signature for sutural stem cellsIn the meningeal layers, we detect a potential chondrogenic periosteal dura population that may be involved in endochondral ossification that closes sutures. Expression of genes mutated in craniosynostosis is spread across diverse cell types, suggesting multiple points at which homeostasis can fail. This single-cell atlas provides a resource to understand the development of the coronal suture, the suture most commonly fused in craniosynostosis.
Project description:Beyond forming bone, osteoblasts play pivotal roles in various biological processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have recently been implicated in intercellular communication via transfer of proteins and nucleic acids between cells. Here, we focused on the proteomic characterization of non-mineralizing (NMOBs) and mineralizing (MOBs) human osteoblast (SV-HFOs) EVs and investigated their effect on human prostate cancer (PC3) cells by microscopic, proteomic and gene expression analyses. Proteomic analysis showed that 97% of the proteins were shared among NMOB and MOB EVs, and 30% were novel osteoblast-specific EV proteins. Label-free quantification demonstrated mineralization stage-dependent five-fold enrichment of 59 and 451 EV proteins in NMOBs and MOBs, respectively. Interestingly, bioinformatic analyses of the osteoblast EV proteomes and EV-regulated prostate cancer gene expression profiles showed that they converged on pathways involved in cell survival and growth. This was verified by in vitro proliferation assays where osteoblast EV uptake led to two-fold increase in PC3 cell growth compared to cell-free culture medium-derived vesicle controls. Our findings elucidate the mineralization stage-specific protein content of osteoblast-secreted EVs, show a novel way by which osteoblasts communicate with prostate cancer, and open up innovative avenues for therapeutic intervention. PC3 cells were treated with extracellular vesicles from non-mineralizing and mineralizing SV-HFOs for three different incubation times (4hrs, 24hrs, 48hr)
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.