Profiling FACS Sorted Calvarial Osteoblast Cultures: Day 7 (pOBCol3.6GFP) and Day 17 (pOBCol2.3GFP)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE2332 | GEO | 2005/04/20
SECONDARY ACCESSION(S): PRJNA91553
REPOSITORIES: GEO
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2007-08-31 | E-GEOD-2332 | biostudies-arrayexpress
Project description:Manzamine-A is a marine-derived alkaloid which has anti-viral and anti-proliferative properties and is currently being investigated for its efficacy in the treatment of certain viruses (malaria, herpes, HIV-1) and cancers (breast, cervical, colorectal). Manzamine-A has been found to exert effects via modulation of SIX1 gene expression, a gene critical to craniofacial development via the WNT, NOTCH, and PI3K/AKT pathways. To date little work has focused on Manzamine-A and how its use may affect bone. We hypothesize that Manzamine-A, through SIX1, alters bone cell activity. Here, we assessed the effects of Manzamine-A on cells that are responsible for the generation of bone, pre-osteoblasts and osteoblasts. PCR, qrtPCR, MTS cell viability, Caspase 3/7, and functional assays were used to test the effects of Manzamine-A on these cells. Our data suggests Six1 is highly expressed in osteoblasts and their progenitors. Further, osteoblast progenitors and osteoblasts exhibit great sensitivity to Manzamine-A treatment exhibited by a significant decrease in cell viability, increase in cellular apoptosis, and decrease in alkaline phosphatase activity. In silico binding experiment showed that manzamine A potential as an inhibitor of cell proliferation and survival proteins, i.e., Iκb, JAK2, AKT, PKC, FAK, and Bcl-2. Overall, our data suggests Manzamine-A may have great effects on bone health overall and may disrupt skeletal development, homeostasis, and repair.
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Project description:BackgroundEnterococcus faecalis is a dominant pathogen in the root canals of teeth with persistent apical periodontitis (PAP), and osteoblast apoptosis contributes to imbalanced bone remodelling in PAP. Here, we investigated the effect of E. faecalis OG1RF on apoptosis in primary human calvarial osteoblasts. Specifically, the expression of apoptosis-related genes and the role of anti-apoptotic and pro-apoptotic members of the BCL-2 family were examined.MethodsPrimary human calvarial osteoblasts were incubated with E. faecalis OG1RF at multiplicities of infection corresponding to infection time points. Flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, caspase-3/-8/-9 activity assay, polymerase chain reaction (PCR) array, and quantitative real-time PCR were used to assess osteoblast apoptosis.ResultsE. faecalis infection increased the number of early- and late-phase apoptotic cells and TUNEL-positive cells, decreased the mitochondrial membrane potential (ΔΨm), and activated the caspase-3/-8/-9 pathway. Moreover, of all 84 apoptosis-related genes in the PCR array, the expression of 16 genes was upregulated and that of four genes was downregulated in the infected osteoblasts. Notably, the mRNA expression of anti-apoptotic BCL2 was downregulated, whereas that of the pro-apoptotic BCL2L11, HRK, BIK, BMF, NOXA, and BECN1 and anti-apoptotic BCL2A1 was upregulated.ConclusionsE. faecalis OG1RF infection triggered apoptosis in human calvarial osteoblasts, and BCL-2 family members acted as regulators of osteoblast apoptosis. Therefore, BCL-2 family members may act as potential therapeutic targets for persistent apical periodontitis.
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Project description:To identify genome-wide, direct targets of HES1 in human pancreas progenitors, we performed Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) of endogenous HES1 in the HUES4 PDX1GFP/+ reporter cell line on Day 13 of differentiation from both unsorted bulk populations and FACS-sorted GFP+ cells.
2022-02-27 | E-MTAB-6922 | biostudies-arrayexpress
Project description:Craniosynostosis is the premature fusion of the sutures of the calvaria and is principally designated as being either syndromic (demonstrating characteristic extracranial malformations) or non-syndromic. While many forms of syndromic craniosynostosis are known to be caused by specific mutations, the genetic etiology of non-syndromic, single-suture craniosynostosis (SSC) is poorly understood. Based on the low recurrence rate (4-7%) and the fact that recurrent mutations have not been identified for most cases of SSC, we propose that some cases of isolated, single suture craniosynostosis may be polygenic. Previous work in our lab identified a disproportionately high number of rare and novel gain-of-function IGF1R variants in patients with SSC as compared to controls. Building upon this result, we used expression array data from calvarial osteoblasts isolated from infants with and without SSC to ascertain correlations between high IGF1 expression and expression of other osteogenic genes of interest. We identified a positive correlation between increased expression of IGF1 and RUNX2, a gene known to cause SSC with increased gene dosage. Subsequent phosphorylation assays revealed that osteoblast cell lines from cases with high IGF1 expression demonstrated inhibition of GSK3?, a serine/threonine kinase known to inhibit RUNX2, thus activating osteogenesis through the IRS1-mediated Akt pathway. With these findings, we have utilized established mouse strains to examine a novel model of polygenic inheritance (a phenotype influenced by more than one gene) of SSC. Compound heterozygous mice with selective disinhibition of RUNX2 and either overexpression of IGF1 or loss of function of GSK3? demonstrated an increase in the frequency and severity of synostosis as compared to mice with the RUNX2 disinhibition alone. These polygenic mouse models reinforce, in-vivo, that the combination of activation of the IGF1 pathway and disinhibition of the RUNX2 pathway leads to an increased risk of developing craniosynostosis and serves as a model of human SSC.
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Project description:We show that high quality microarray gene expression profiles can be obtained following FACS sorting of cells using combinations of transcription factors. We use this transcription factor FACS (tfFACS) methodology to perform a genomic analysis of hESC-derived endodermal lineages marked by combinations of SOX17, GATA4, and CXCR4, and find that triple positive cells have a much stronger definitive endoderm signature than other combinations of these markers. Additionally, SOX17+GATA4+ cells can be obtained at a much earlier stage of differentiation, prior to expression of CXCR4+ cells, providing an important new tool to isolate this earlier definitive endoderm subtype. Overall, tfFACS represents an advancement in FACS technology which broadly crosses multiple disciplines, most notably in regenerative medicine to redefine cellular populations. 21 Total samples were analyzed. Samples collected after 5 days of hESC differentiation included SOX17+GATA4+ CXCR4+ cells, unfixed CXCR4+ cells, and unsorted fixed cells. Samples collected after 3 days of differentiation included SOX17+GATA4+ cells, SOX17-GATA- cells, and unsorted fixed cells. As controls, we analyzed both unfixed hESCs and fixed hESCs. All samples contained biological duplicates, triplicates or quadriplicates. We performed hierarchical clustering to demonstrate whether cellular fixation alone could change gene expression. We based this analysis on 1647 transcript clusters with coefficient of variation > 0.5 across the samples and expression values >= 500 in at least 2 out of the 21 samples. We found that fixation and staining steps did not cause distortions in gene expression measurements. This is supported by the fact that fixed and unfixed cells cluster together based upon differentiation stage, not based upon degree of fixation. Furthermore, using GSEA analysis, we found that the SOX17+GATA4+CXCR4+ day 5 cells and day 3 SOX17+GATA4+ were more enriched for compiled gold-standard endodermal genes than the CXCR4+ day 5 population.
2011-08-23 | E-GEOD-24135 | biostudies-arrayexpress
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| 2341570 | ecrin-mdr-crc
Project description:BackgroundMechanical therapies, such as distraction osteogenesis, are widely used in dental clinics. During this process, the mechanisms by which tensile force triggers bone formation remain of interest. Herein, we investigated the influence of cyclic tensile stress on osteoblasts and identified the involvement of ERK1/2 and STAT3.Materials and methodsRat clavarial osteoblasts were subjected to tensile loading (10% elongation, 0.5 Hz) for different time periods. RNA and protein levels of osteogenic markers were determined using qPCR and western blot after inhibition of ERK1/2 and STAT3. ALP activity and ARS staining revealed osteoblast mineralization capacity. The interaction between ERK1/2 and STAT3 was investigated by immunofluorescence, western blot, and Co-IP.ResultsThe results showed that tensile loading significantly promoted osteogenesis-related genes, proteins and mineralized nodules. In loading-induced osteoblasts, inhibition of ERK1/2 or STAT3 decreased osteogenesis-related biomarkers significantly. Moreover, ERK1/2 inhibition suppressed STAT3 phosphorylation, and STAT3 inhibition disrupted the nuclear translocation of pERK1/2 induced by tensile loading. In the non-loading environment, inhibition of ERK1/2 hindered osteoblast differentiation and mineralization, while STAT3 phosphorylation was elevated after ERK1/2 inhibition. STAT3 inhibition also increased ERK1/2 phosphorylation, but did not significantly affect osteogenesis-related factors.ConclusionTaken together, these data suggested that ERK1/2 and STAT3 interacted in osteoblasts. ERK1/2-STAT3 were sequentially activated by tensile force loading, and both affected osteogenesis during the process.
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Project description:We differentiated mouse Col2.3 GFP ESCs and iPSCs to osteoblast progenitors and confirmed the osteoblast identity of ESC- and iPSC-derived osteoblasts using RNA-sequencing and hierarchical clustering, and demonstrate that Col2.3GFP+ sorted cells are more similar to freshly isolated Col2.3GFP+ osteoblasts from mouse bone than to unsorted cells based on whole genome expression profiling.
2018-05-01 | GSE110434 | GEO