Gene expression profiling of skeletal myoblast differentiation and exposure to TNF-alpha or IGF1
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Swanhild Meyer
PROVIDER: E-MTAB-3474 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Murine skeletal muscle cells pmi28 were cultured in growth medium (HamM-^Rs F10, supplemented with 20 % FCS, 2 mM L-glutamine, and 1 % Penicillin / Streptomycin). Myoblasts were cultured on laminin-1 coated dishes for an additional 24 h before switching a fraction of dishes to differentiation medium (DMEM medium containing 2 % horse serum, 2 mM L glutamine, and 1 % Penicillin / Streptomycin) and to differentiation medium containing either 2 x 103 U/ml mouse recombinant TNF-? (Roche Applied Science), or 5 M-5g/ml mouse recombinant IGF-I (Sigma-Aldrich), or carrier only. pmi28 cells (TNF-? and IGF-I treatments as well as controls) were harvested 24 h after the induction of differentiation. The experiments were performed in quadruplicates or triplicates, respectively.
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Project description:Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current skeletal-muscle-models in vitro are incapable of fully recapitulating its physiological functions especially regarding exercise. Supplementation of IGF1, a growth factor secreted by myofibers in vivo, might help to overcome these limitations. Primary human CD56-positive myoblasts were differentiated into myotubes in the presence/absence of IGF1 in serum-free medium for 10 days. Daily collected samples were analyzed by proteomics, qRT-PCR and myotube contractibility via electrical-pulse-stimulation (EPS). Mitochondrial respiration and glucose uptake were measured. IGF1-supported differentiation formed thicker multinucleated myotubes showing physiological contraction upon EPS following day 6 while myotubes without IGF1 were almost incapable of contraction. IGF1-treatment upregulated particularly muscle-specific proteins that contribute to myofibril and sarcomere assembly, striated muscle contraction, and ATP production. Elevated PPARGC1A, MYH7 and reduced MYH1/2 suggest a switch towards a more oxidative phenotype in line with elevated mitochondrial respiration. IGF1-treatment also upregulated expression of GLUT4 and increased insulin-dependent glucose uptake. To conclude, utilizing IGF1, we engineered human myotubes that recapitulate the physiological traits of skeletal muscle in vivo vastly superior to established protocols. This novel model enables investigation of exercise on a molecular level, drug screening and interorgan-crosstalk by transfer to organ-on-chip.
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Project description:All established protocols for differentiation of mouse and human pluripotent stem cells into specific neural subpopulations generate a considerable cellular heterogeneity that hampers experimental and clinical progress. In order to obtain a homogenous population of neuronal precursor cells and to streamline the differentiation of embryonic stem cells (ESCs), we assessed PSA-NCAM, a surface glycoprotein that is specifically expressed on immature neurons. We developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from differentiated ESC cultures and characterized their neuronal differentiation potential in vitro. PSA-NCAM enrichment at an early step of neural differentiation increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. The in vivo potential of in vitro derived PSA-NCAM+ enriched precursors was functionally characterized by grafting into the forebrain of adult mice. Analysis for several neuronal and glia markers at 10 or 40 days post graft showed a distinct differentiation pattern. While unsorted control cells gave rise to a mixed population composed of immature precursors, early postmitotic neurons or glial cells, the majority of PSA-NCAM+ enriched cells differentiated into NeuN positive neurons. Furthermore, when in contact with the rostral migratory stream, higher numbers of cells integrated into the stream and migrated towards the olfactory bulb when the PSA-NCAM enriched population was grafted. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells. Two conditions (step 4, step 5), each represented by three biological replicates of control and enriched cells (Cy5); mESC was used as common reference (Cy3)
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Project description:The treatment of bone defects caused by infection, trauma or neoplasms remains a clinical challenge. Autologous bone transplantation is limited by availability, donor site morbidity and surgical risk factors. This has given rise to stromal/stem-cell based therapy. Bone marrow derived stromal cells (BMSCs) have been studied to a large extent and show high regenerative potential but their use is limited by availability, donor site morbidity and the relatively low cell yield as they represent only <0.1% of cell harvested from bone marrow aspirate. At the same time, they are the closest mesenchymal stromal cells for bone tissue engineering given their tissue origin and, unlike other mesenchymal stromal cells, can support the formation of hematopoietic marrow. Adipose tissue derived stromal cells (ASCs) as part of the stromal vascular fraction of adipose tissue can as well undergo osteogenic differentiation but can be additionally isolated in a sufficient quantity from lipoaspirate after liposuction of abundant subcutaneous fat tissue. Here, it has been shown that there are no major differences in regard to proliferation or differentiation capacity of ASCs derived from subcutaneous fat of different anatomical regions. It has been shown that BMSCs are more prone to senescence during expansion and passage than ASCs and that ageing impacts proliferative capabilities of BMSCs more than that of ASCs while it has also been reported that osteogenic differentiation capacity is least impacted by age. Multiple studies have compared the characteristics of these two mesenchymal stromal cells in regard to bone tissue engineering in vitro. Most studies point to inferior extracellular matrix mineralization and lower expression of key osteogenic transcription markers like Runx2 in osteogenic differentiated ASCs compared to BMSCs. On the other hand, a study by Rath et al. found contrary results using particular culturing conditions like 3D bioglass scaffolds. An intraindividual comparison of human MSCs of three donors cultured on decellularized porcine bone confirmed superior osteogenic capacity of BMSCs compared to ASCs. In contrast to BMSCs, ASCs were not able to induce heterogenic ossification in a mouse model. In a sheep tibia defect model application of BMSCs resulted in a significantly higher amount of newly formed bone tissue. Importantly, Osteogenic differentiated ASCs do not support the formation of a hematopoietic marrow. Proteomics enables large-scale analysis of proteins present in a cell type and can be used to identify differentially regulated key proteins in a comparative approach. A comparative proteomic analysis of BMSCs and ASCs by Roche et al. in 2009 identified 556 proteins with 78% of these not being differentially regulated between these two cell populations, regarded as high similarity. Another comparative proteomic study of 2016 by Jeon et al. found 90 differentially regulated proteins out of 3000 total identified proteins. Both studies do not specify a number of different tissue donors and in part using cell lines. Looking for differences upon osteogenic differentiation, transcriptomic comparison of osteogenic differentiated porcine ASCs and BMSCs has been performed, resulting in 21 differentially expressed genes after 21 days of osteogenic culture conditions. Still, it remains unanswered, which are the key distinctive features of osteogenic differentiated ASCs and BMSCs at protein level that might help address the abovementioned weaknesses of ASCs in bone tissue engineering/regeneration for translational research. To overcome this need, an intraindividual comparative DIA based proteomic analysis of osteogenic differentiated human BMSC and ASCs was performed in this study.
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Project description:We analysed gene expression profiles in dental follicle cells after 7 days of osteogenic differentiation with different inducers. Total RNAs were isolated from dental follicle cells after 7 days of differentiation with dexamethasone, BMP2, IGF2 and for control with standard cell culture medium
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Project description:Carbon monoxide (CO) abrogates TNF-alpha mediated inflammatory responses in endothelial cells, yet, the underlying mechanism hereof is still elusive. We sought to explore potential mechanisms by which CO down-regulates VCAM-1 expression on TNF-alpha stimulated human umbilical vein endothelial cells (HUVEC). By genome-wide gene expression profiling and pathway analysis we studied the relevance of particular pathways for the anti-inflammatory effect of CO. In CO-releasing molecules-3 (CORM-3) stimulated HUVEC, significant changes in gene expression were found for genes implicated in the proteasome and porphyrine pathways. Although proteasome activities were increased by CORM-3, proteasome inhibitors did not abolish CORM-3‘s effect. Likewise, HO-1 inhibitors did not abrogate the ability of CORM-3 to down-regulate VCAM-1 expression. MAPK p38 was inhibited by CORM-3. Accordingly, VCAM-1 expression was down-regulated by the p38 inhibitor SB203580. Down-regulation of VCAM-1 by CORM-3 only occurred at concentrations that partly inhibit ATP production. Sodium azide and oligomycin paralleled the effect of CORM-3 in this regard. In conclusion, down-regulation of VCAM-1 by CORM-3 seems to be mediated via inhibition of p38 and mitochondrial respiration. Although CORM-3 up-regulates several genes in the ubiquitin proteasome sytem (UPS) or porphyrin pathway, there is no evidence that these changes are involved in the anti-inflammatory properties of CORM-3. In this study we focused on the potential of CORM-3 to downregulate the expression of TNF-alpha mediated inflammataroy genes
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Project description:In this work we present an analytical strategy to systematically identify early regulators by combining gene regulatory networks (GRN) with GWAS. We hypothesized that early regulators in T-cell associated diseases could be found by defining upstream transcription factors (TFs) in T-cell differentiation. Time series expression and DNA methylation profiling of T-cell differentiation identified several upstream TFs, of which TFs involved in Th1/2 differentiation were most enriched for disease associated SNPs identified by GWAS. Naïve CD4+ T cells were isolated from buffy coat of four healthy donors using a naïve CD4+ T cell isolation kit (Miltenyi, Bergisch-Gladbach, Germany). Naïve CD4+ T cells were stimulated with TGF-β (1 ng/mL), IL-1β (10 ng/mL), IL-6 (25 ng/mL), IL-21 (25 ng/mL) and IL-23 (25 ng/mL) for Th17, and TGF-β (10 ng/mL) and IL-2 (10 ng/mL) for Treg. Cells were cultured for six days in Iscoveâs modified Dulbecco medium (IMDM) supplemented with 2 mM L-glutamine (PAA Laboratories, Linz, Austria), 10% heat-inactivated FCS (PAA Laboratories, Linz, Austria), 5 µM βâmercaptoethanol (Sigma-Aldrich, St. Louis, Missouri, USA) and 50 ug/mL gentamicin (Sigma-Aldrich, St. Louis, Missouri, USA). Cells were cultured for 6 days and then re-stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of corresponding polarizing cytokines and antibodies for another 2 days (Zhang et al. 2013). RNA was extracted using a miRneasy Mini Kit (Qiagen). The RNA concentrations were analysed with NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). For the gene expression microarray analysis, The cRNA was prepared using a Low Input QuickAmp Labeling Kit. For Th17 and Treg cells the gene expression microarray analysis was performed using SurePrint G3 Human Gene Expression 8x60K v2 microarray kit, according to the manufactureâs instruction (Agilent Technologies).
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