Microarray gene expression profiles of adipose-derived mesenchymal stem cells from LEW and PVG rats upon 24h and 48h LPS stimulation
ABSTRACT: We have extended our investigation to differential immunogenicity between tolerogenic PVG rats and immunogenic LEW rats by analyzing gene expression in adipose-derived mesenchymal stem cells (ASCs) with LPS stimulation. Furthermore, to establish a direct link between gene expression and immunogenic functional annotation, ASCs from LEW and PVG rats were obtained, and the effects of inherent difference and LPS treatment on global gene expression were evaluated using microarray analyses. We analyzed microarray gene expression profiles of ASCs from 3 LEW and 3 PVG rats of 8-week age and separated ASCs from each individual into four conditions, 24h and 48h control, 24h and 48h with 1 μg/ml LPS stimulation. The total RNA samples of triplicate ASCs isolated from LEW and PVG with and without LPS treatment were pooled in each condition and the global gene expression profiles were analyzed using microarray.
Project description:We have extended our investigation to differential immunogenicity between tolerogenic PVG rats and immunogenic LEW rats by analyzing gene expression in adipose-derived mesenchymal stem cells (ASCs) with LPS stimulation. Furthermore, to establish a direct link between gene expression and immunogenic functional annotation, ASCs from LEW and PVG rats were obtained, and the effects of inherent difference and LPS treatment on global gene expression were evaluated using microarray analyses. Overall design: We analyzed microarray gene expression profiles of ASCs from 3 LEW and 3 PVG rats of 8-week age and separated ASCs from each individual into four conditions, 24h and 48h control, 24h and 48h with 1 μg/ml LPS stimulation. The total RNA samples of triplicate ASCs isolated from LEW and PVG with and without LPS treatment were pooled in each condition and the global gene expression profiles were analyzed using microarray.
Project description:Using a rat orthotopic liver transplantation (OLT) model and microarray, we compared the expression profiles of microRNAs in naïve and AR livers at day 7 after OLT obtained from the rats with short-term (<14 days, DA-LEW) or long-term (>60 days, DA-PVG) survival fate. AR-related microRNAs and pro-inflammatory cytokines were validated by using a quantitative real-time PCR. AR-related microRNA-mediated inflammatory responses were evaluated by overexpression of a target microRNA in rat primary hepatocytes. Human liver biopsies from recipients with AR or abnormal liver function were used for clinical verification. The microarray revealed that miR-301a was significantly upregulated in lethal AR livers of DA-LEW, while it was comparable to the naïve livers in DA-PVG, which spontaneously overcomes AR. OLT was carried out following the technique previously described by Kamada et al. in the following combinations: DA donor liver into PVG (DA-PVG; natural tolerance model with long-term (>60 days) and DA donor liver into LEW (DA-LEW; AR model with short-term (<14 days).
Project description:CD14+ Monocytes from healthy volunteers were purified by MACS (negative selection) and FACSorting and either left untreated or stimulated for 24h and 48h with LPS. THP-1 cells were stimulated for 4h, 24h and 48h with LPS. Glycoproteins were captured with hydrazide chemistry and tryptic and PNGase F-released peptide fractions analyzed by MS/MS. Quantitative assessment revealed differential glycoprotein expression in activated/LPS-tolerized monocytes and naïve monocytes and THP-1 cells.
Project description:Adipose tissue-derived stromal stem cells (ASCs) represent a promising regenerative resource for soft tissue reconstruction. To understand the changes in cell function during the transition of ASCs into fully mature fat cells, we compared the transcriptome profiles of cultured undifferentiated human primary ASCs under conditions leading to acquisition of a mature adipocyte phenotype by microarray analysis. Microarray analysis was performed on total RNA extracted from separate ACS isolates of six human adult females before and after 7 days (7d: early stage) and 21 days (21d: late stage) of adipocyte differentiation in vitro.
Project description:We have previously reported that the deficiency of p53 alone or in combination with Rb (Rb-/- p53-/-) in adipose-derived MSCs (ASCs) promotes leiomyosarcoma-like tumors in vivo. Here, we hypothesized that the source of MSCs and/or the cell differentiation stage could determine the phenotype of sarcoma development. To investigate whether there is a link between the source of MSCs and sarcoma phenotype, we generated p53-/- and Rb-/-p53-/- MSCs from bone marrow (BM-MSCs). Both genotypes of BM-MSCs initiated leiomyosarcoma formation similar to p53-/- and Rb-/-p53-/- ASCs. In addition, gene expression profiling revealed a link between p53- or Rb-p53-deficient BM-MSCs and ASCs and muscle-associated sarcomagenesis. These data suggest that the tissue source of MSC does not seem a crucial factor in the development of a particular sarcoma phenotype. To analyze whether the differentiation stage defines the sarcoma phenotype, BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage, and both p53 and Rb were excised using Cre-expressing adenovectors at different stages along osteogenic differentiation. Regardless of the level of osteogenic commitment, the inactivation of Rb and p53 in BM-MSC-derived, but not in ASC-derived, osteogenic progenitors gave rise to osteosarcoma-like tumors which could be serially transplanted. This indicates that the osteogenic differentiation stage of BM-MSCs imposes the phenotype of in vivo sarcoma development, and that BM-MSC-derived osteogenic progenitors rather than undifferentiated BM-MSCs, undifferentiated ASCs or ASC-derived osteogenic progenitors, represent the cell of origin for osteosarcoma development. To analyse whether the BM-MSC and Fat-MSC (ASC) differentiation stage may define the sarcoma phenotype, RbloxP/loxPp53loxP/loxP BM-MSCs and ASCs were induced to differentiate towards the osteogenic lineage and both Rb and p53 were excised with adenoviral vectors expressing the Cre-recombinase gene (Ad-CMV-Cre) at different stages (day 0 and 10) along osteogenic differentiation. NSG mice were inoculated subcutaneously with 5×10^6 mutant cells. Animals were killed when tumors reached 1 cm3 or 150 days after infusion. Some of the obtained tumors were mechanically disaggregated to establish ex vivo MSC-transformed cell lines. Gene expression analysis was performed using: WT BM-MSCs and ASCs, Rb-/-p53-/- BM-MSCs and ASCs previously differentiated to the osteogenic lineage for 10 days and a tumor cell line derived from p53-/-Rb-/- BM-MSC differentiated to the osteogenic lineage for 10 days.
Project description:The effect of the GSK3 inhibitor Azakenpaullone (Azak) and the differentiation agent retinoic acid (RA) was studied in low and high MYCN levels in the MYCN inducible neuroblastoma cell line SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN (SY5Y-MYCN). Azak was dissolved in DMSO in order to apply it to the cells. Therefore a vehicle control consisting of SY5Y-MYCN cells treated with 24h 1 ul/ml DMSO only was used in duplicates. Doxycycline (Sigma) dissolved in water was used at a final concentration of 1ug/ml to induce MYCN expression in SY5Y-MYCN. A co-treatment study with Dox and Azak was conducted. SY5Y-MYCN cells were treated with 24h Azak, 24h Azak & 48h Dox and 48h Dox, with biological duplicates. 1 uM RA (dissolved in DMSO) and 1 ug/mL Doxycycline were given individually and in combination. SY5Y-MYCN cells were treated with 24h RA, 24h RA & 48h Dox, and 48h Dox and RNA was extracted in biological duplicates. For the 24h RA & 48h Dox co-treatment cells were treated with Dox for 24h and then with RA and fresh Dox for a further 24h.
Project description:Adipose derived stem cells (ASCs) were preconditioned with endothelial differentiation medium (EDM) for four days and then incubated in endothelial basal medium (EBM) supplemented with 1% microvesicle (MV)-free FBS for two days. The conditioned medium was harvested for MV isolation. The small RNA extracted from the MVs was used for microRNA array. qPCR gene expression profiling; Two equal-amount small RNA samples were from two independent experiments.
Project description:Stimulation of estrogen receptor beta in PHPT, genetic changes after 24 and 48h of treatments vs. Control Treatment of parathyroid adenomas (4 patients, 4 adenomas) with DPN 24h (4 samples), DPN 48h (4 samples), OHT 24h (4 samples), OHT 48h (4 samples), control 24h (3 samples), control 48h (4 samples). Omission of 1 sample based on low RNA quality.
Project description:RasGRP4 is required for CD117+ DCs to optimally induce certain NK cell-dependent immune responses in vivo and in vitro in response to LPSRasGRP4 expressed in DCs played an important role on NK cell IFN-γ secretion . We used microarrays to detail the global programme of gene expression underlying cellularisation and identified distinct classes of up-regulated or down-regulated genes between the two DCs from WT and RasGRP4 mice respectively upon LPS stimulation. CD117+splenocytes were extracted from WT and RasGRP4 KO mice spleens aged 9-12 weeks and plated in 6-well plates with 1ug/mL of LPS for 0h,6h,12h,24h and 48h repectively for RNA extraction and hybridization on Affymetrix microarrays. Time course.