In vitro assessment of drug-induced liver steatosis based on human dermal stem cell-derived hepatic cells.
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ABSTRACT: Human skin-derived precursor cells (hSKP) are a post natal stem cell population isolated from the dermis. These cells acquire hepatic characteristics upon differentiation with hepatogenic factors. Differentiated hSKP show characteristics of hepatocyte precursor cells and respond to hepatotoxic compounds in a comparable way as human hepatocyte cultures. We used microarray analyses to evaluate the modulation of gene expression due to exposure to a steatosis-inducing compound. hSKP obtained from 3 different donors were firstly differentiated and cultivated in the presence or absence of sodium valproate (Na-VPA). Control and Na-VPA samples were collected after 24h exposure.
Project description:Human skin-derived precursor cells (hSKP) are a stem cell population that represents key candidates for cell based-therapy. Inflammation, however, is often present in situations where cellular replacement therapy is required. These inflammatory conditions, and more specifically the presence of the cytokine interferon (IFN)-M-NM-3, might result in an increase of MHC class II antigens in hSKP-derived grafts and facilitate their rejection. We used microarray analyses to confirm the activated status of the pro-inflammatory condition, induced by exposure to a cocktail of pro-inflammatory cytokines (IL-1M-NM-2, IFN-M-NM-3, TNFM-NM-1 and IFN-M-NM-1). Consequently, we investigated the modulation by inflammation of canonical pathways related to immunology in hSKP. hSKP obtained from 3 different donors (passage 4) were cultivated in the presence or absence of the pro-inflammatory cocktail. Control and pro-inflammatory stimulated samples were collected 18h after treatment.
Project description:We used gene expression accompanied by physical characteristics and gill Na+/K+-ATPase activity to analyze physiological differences associated with two life history variations of juvenile fall Chinook Salmon in the Snake River basin. Subyearlings originating in the Snake River typically migrate seaward as subyearlings, whereas many subyearlings from the Clearwater River delay seaward migration during summer and complete seaward migration the following spring as yearlings. We examined gill Na+/K+-ATPase activity and gene expression of subyearlings at different times during rearing and seaward emigration. Natural-origin Snake River subyearlings rearing under an increasing photoperiod and seasonally increasing temperatures showed a typical increasing pattern of parr to smolt gill Na+/K+-ATPase activity development, which then declined into autumn. In contrast, Clearwater River subyearlings that had experienced cooler temperatures showed no pattern of increasing gill Na+/K+-ATPase activities and were not different from parr. Liver transcription of genes involved in DNA repair and binding, the cell cycle, metabolism (steroid, fatty acid and other metabolic pathways) iron homeostasis, heme and oxygen binding, the immune response, and male sexual development were enriched amongst genes differentially expressed between Snake River parr versus smolts. Gene expression results confirmed that Clearwater River subyearlings were parr-like in their physiological status. By autumn, subyearlings had low gill Na+/K+-ATPase activities despite their large size and external smolt characteristics. We suggest that environmental factors like temperature and photoperiod influence subyearling physiological status in each river that ultimately dictates juvenile life history pathways. Non-migrating and migrating natural subyearling fall Chinook salmon were collected from the Snake River. Non-migrating natural subyearling fall Chinook salmon were collected from the Clearwater River. Twelve fish were collected at each of four different time points for a total of 48 fish. Total RNA was extracted from the liver of each fish. Equal amounts of RNA from three fish were pooled to create four pools of RNA per time point. Each RNA pool was hybridized to an array for a total of 16 arrays with four arrays per time point.
Project description:Histone deacetylase (HDAC) inhibitors are widely utilized in hematopoietic malignance therapy; nevertheless, little is currently known concerning their effects on normal myelopoiesis. In order to investigate a putative interference of HDAC inhibitors in myeloid commitment of hematopoietic stem/progenitor cells (HSPCs) we treated CD34+ cells with valproic acid (VPA). Moreover, we investigate changes in gene expression induced by VPA treatment on HSPCs, by means of microarray analysis in VPA treated and untreated (CTR) CD34+ cells. VPA treatment induced H4 histone acetylation in CD34+ cells and blocked them in G0-G1 phase of cell cycle. CD34 expression is maintained for a longer time in VPA treated cells, while the physiological decrease of CD34 antigen occurred in CTR cells. Moreover, VPA favored erythrocyte and megakaryocyte differentiation at the expense of granulocyte and mono-macrophage lineages, as demonstrated by immunophenotyping, morphological and clonogenic analysis. Finally, we demonstrated that VPA up-regulated master gene regulators of erythrocyte and megakaryocyte differentiation (GFI1B and MLLT3) through histone iper-acetylation of their promoters. These results indicate that VPA treatment enhances erythrocyte and megakaryocyte differentiation at the expense of granulocyte and mono-macrophage one. Microarray data provide for the first time a detailed molecular support for the biological effects promoted by VPA treatment in HSPCs. Human CD34+ cells were purified from umbilical Cord Blood (CB) samples. After an initial 24 hours of incubation, CD34+ cells were exposed to VPA. Total cellular RNA was extracted from untreated (CTR) and VPA treated CD34+ HSCs after 48 hours of treatment.
Project description:Rat Extraembryonic Endoderm Precursor (XEN-P) cells resemble the nascent hypoblast of the blastocyst. Here we show that rat Multipotent Adult Progenitor Cell (rMAPC) lines derived from bone marrow (BM) exhibit salient nascent hypoblast characteristics. We could not identify cells with hypoblast features in rat BM, but they appeared after prolonged in vitro culture under rMAPC conditions. However, rMAPC culture conditions supported the very rapid isolation of new lines with nascent hypoblast features from rat blastocysts that were more homogenous than XEN-P cells isolated on feeders. The gene expression profile, growth factor requirement, Oct4 promoter methylation, and in vitro/vivo differentiation potential of cells isolated using rMAPC culture conditions from BM or blastocysts or XEN-P cells were highly similar. We conclude that the three cell populations have characteristics of hypoblast stem cells (HypoSC) and that rMAPC culture conditions support the isolation of XEN-P/HypoSC from the blastocyst and induction of HypoSC from BM. Comparison between bone marrow and blastocyst-derived Hypoblast Stem Cells (hypoSC) Three cell lines derived from bone marrow, three cell lines derived from blastocyst and one mesenchymal used, as control (for all of them 2 replicates)
Project description:Sirtuins, a family of histone deacetylases, have a fiercely debated role in regulating lifespan of different species. Contrasting recent observations, we here find that overexpression of sir-2.1, the orthologue of mammalian SirT1, does extend C. elegans lifespan. Sirtuins are known to convert NAD+ into nicotinamide (NAM). We here find that NAM and its metabolite, 1-methylnicotinamide (MNA), extend C. elegans lifespan, also in the absence of sir-2.1. Consistently, impairment of sir-2.1 prevents extension of lifespan by nicotinic acid (NA), a NAD+ precursor. Taken together, sirtuins extend lifespan by promoting formation of MNA to generate a phase I - mediated ROS signal, providing an unexpected mechanistic role for sirtuins beyond histone deacetylation. 9 samples: 3 mRNA profiles of C.elegans 48h after L4 exposed to nicotinic acid; 3 mRNA profiles of C.elegans 48h after L4 exposed to 1-MNA; 3 mRNA profiles of C.elegans 48h after L4 as controls (H20)
Project description:Genome-wide maps of the H3K9 acetylation state in embryonic stem cells (ESCs) before and after treatment with low levels of the histone deacetylase (HDAC) inhibitor valproic acid (VPA). ChIP-seq for 3 samples: untreated E14 cells, cells treated with VPA for 4 hrs and cells treated with VPA for 16 hrs. Unprecipitated DNA was used as the input control (Input).
Project description:To develop an in vitro model for developmental toxicity testing, we characterized gene expression changes during mouse embryonic stem cell (mESC) differentiation and their modulation by developmental toxicants. C57BL/6 mESCs were allowed to differentiate spontaneously and global gene expression changes were studied using microarrays at at different time points after embryoid body (EB) formation. Develpmental toxicants tested include 2.0 mM monobutyl phthalate (MBP), 0.25 mM thalidomide (THD), and 1.0 mM valproic acid (VPA). RNA of control samples (unexposed) was collected at 0, 24, 48, 72, 96, 120 and 168 h after EB formation; RNA of compound-exposed samples were collected at 24 h after EB formation. Three replicates were included for each experimental condition.
Project description:Having found that LexA degradation was significantly higher under apoptotic like death (ALD) than under SOS conditions, we hypothesized that additional genes tightly regulated by LexA would be transcribed under ALD conditions. We used microarray analysis to test the transcription pattern of up-regulated genes in ALD. We added no NA (n.t.), 100 M-BM-5g/ml NA, or 10 M-BM-5g/ml NA to cultures of the WT MC4100relA+, or its M-NM-^TmazEF or M-NM-^TmazEFlexA3 derivatives.
Project description:Human skin-derived precursor cells (hSKP) are a post natal stem cell population isolated from the dermis. These cells acquire hepatic characteristics upon differentiation with hepatogenic factors. Differentiated hSKP show characteristics of hepatocyte precursor cells and respond to hepatotoxic compounds in a comparable way as human hepatocyte cultures. We used microarray analyses to evaluate the modulation of gene expression due to exposure to a steatosis-inducing compound.
Project description:Rett syndrome (RTT) is a devastating neurodevelopmental disorder that occurs once in every 10,000-15,000 live female births. Despite intensive research, no effective cure is yet available. Valproic acid (VPA) has been used widely to treat mood disorder, epilepsy, and a growing number of other disorders. In limited clinical studies, VPA has also been used to control seizure in RTT patients with promising albeit somewhat unclear efficacy. In this study we tested the effect of VPA on the neurological symptoms of RTT and discovered that short-term VPA treatment during the symptomatic period could reduce neurological symptoms in RTT mice. We found that VPA restores the expression of a subset of genes in RTT mouse brains, and these genes clustered in neurological disease and developmental disorder networks. Our data suggest that VPA could be used as a drug to alleviate RTT symptoms. Wild type or MeCP2KO mice received daily injections of VPA (300 mg/kg) for 2 weeks. Each experimental condition: WT control, KO treated with VPA (KO+VPA), and KO treated with saline (KO+saline). Half brain samples were retrieved.