Transcriptome analysis of human umbilical cord fibroblasts from babies whose mother experienced preeclampsia
ABSTRACT: In pregnancies involving preeclampsia (PE), there is evidence that the fetal-placental unit is under oxidative stress. Here we examined primary cell lines generated from umbilical cords (UC) delivered by mothers who had either a normal pregnancy or experienced early onset PE to determine whether the two had distinguishable phenotypes. While all UC provided outgrowths when established in 4 % O2, success was less assured for PE cords under ambient (20 % O2) conditions (P < 0.05). Moreover, proliferation rates of established PE lines, although similar to controls in 4 % O2, were significantly lower in 20 % O2. PE lines grown in 4 % O2 were also more susceptible to the pro-oxidant diethylmaleate than control lines, and unlike controls, were not protected by glutathione. Transcriptome profiling revealed only a few differentially regulated genes between PE lines and controls in 4 % O2 conditions, but confirmed the more severely stressed phenotype of the PE lines under 20 % O2. We conclude that the primary UC cell lines generated from PE births maintain a susceptibility to oxidative stress that is stable over many cell divisions, but whether the basis of this vulnerability is genetic or epigenetic remains unclear. RNA was isolated from early passages (< p5) of UC fibroblast lines (15 PE and 9 CTL lines, grown in T25 flasks) when they reached ~90% confluency in 4 % O2 conditions. In order to collect RNA from cells under 20 % O2, cell lines at either p 4, 5, or 6 were switched from 4 % O2 conditions to 20 % O2 conditions when they were approximately 20 % confluent. When they reached ~90% confluency (generally 2 days in 4 % O2 and 3-4 days in 20 % O2 conditions), medium was removed and RNA STAT60 (I ml; Tel-Test, Friendswood, TX) was immediately added to each T25 flask and RNA extracted by following the manufacturer’s instructions. The samples of RNA were submitted to University Texas Southwestern Medical Center Microarray Core Facility (https://microarray.swmed.edu/) and microarray analysis performed with Illumina HumanHT-12 v4 expression BeadChips. Raw intensity data were background subtracted by using BeadStudio software and analyzed further by GeneSpring 12.6 software (Agilent Technologies Inc., Santa Clara CA), according to the advanced workflow protocol: percentile shift and filter by flags (detected).
Project description:The goal of this study was to identify those genes that were differentially expressed in colon cancer cells with mutant K-ras under hypoxic conditions DKS-5 (K-ras mutant) and DKO-3 (K-ras wild-type) were cultured in normoxic (21% O2) and 12-hour hypoxic (1% O2) conditions. This was repeated for a total of 2 samples per condition.
Project description:Transcriptomic analyses of the oxygen response of two glioma cell lines at 20% versus 0.3% O2, and 3% vs 0.3% O2 in the presence or absence of serum 22 samples are analysed with three or more biological replicates in different O2 concentrations.
Project description:Analyze gene expression levels in primary trophoblasts, derived from term human placenta and cultured under standard or hypoxic conditions Human placental trophoblasts were dispersed using a trypsin-deoxyribonuclease-dispase/Percoll method, plated in 6-well plates, and maintained in standard culture conditions (O2=20%). After 4 h (defined as time 0), the plates were divided to those in continued standard culture conditions, or to culture in hypoxia (O2=0%). Cells were then harvested at 6 h, 12 h, 24 h, 48 h and 72 h, and processed for mRNA arrays
Project description:Analyze miRNA expression levels in primary trophoblasts, derived from term human placenta and cultured under standard or hypoxic conditions Human placental trophoblasts were dispersed using a trypsin-deoxyribonuclease-dispase/Percoll method, plated in 6-well plates, and maintained in standard culture conditions (O2=20%). After 4 h (defined as time 0), the plates were divided to those in continued standard culture conditions, or to culture in hypoxia (O2=0%). Cells were then harvested at 6 h, 12 h, 24 h, 48 h and 72 h, and processed for miRNA arrays
Project description:We compared microRNA (miRNA) expression in Ercc1-/- primary mouse embryonic fibroblasts (MEFs) and wild-type (WT) MEFs in 20% O2 and 3% O2 growth conditions to identify miRNAs that drive cellular senescence. MicroRNA expression in WT and Erccc1-/- MEFs was measured at passage 3 and passage 7 after growth in 20% and 3% O2. Two independent experiments were performed for each growth condition.
Project description:Analysis of 5-hydroxymethylcytosine changes in hypoxia in neuroblastoma cells 5-hmC distribution was analyzed from cells exposed to hypoxia (1% O2) or normoxia (20% O2); mRNA was sequenced in parallel for expression analysis.
Project description:Hypoxia plays a key pathogenic role in the outcome of many pathologic conditions. To elucidate how organisms successfully adapt to hypoxia, a population of Drosophila melanogaster was generated, through an iterative selection process, that is able to complete its lifecycle at 4% O2, a level lethal to the starting parental population. Transcriptomic analysis of flies adapted for >200 generations was performed to identify pathways and processes that contribute to the adapted phenotype, comparing gene expression of three developmental stages with generation-matched control flies. A third group was included, hypoxia-adapted flies reverted to 21% O2 for five generations, to address the relative contributions of genetics and hypoxic environment to the gene expression differences. We identified the largest number of expression differences in 0.5-3 hr post-eclosion adult flies that were hypoxia-adapted and maintained in 4% O2, and found evidence that changes in Wnt signaling contribute to hypoxia tolerance in flies. A population of flies able to complete their life cycle at 4% O2 was selected from a starting population of 27 isogenic D. melanogaster lines exposed to increasingly lower O2 levels over many generations. Transcriptomic analysis of adapted flies maintained at 4% O2 or reverted to room air for five generations, and of generation matched naive controls, was performed to better understand changes in gene expression in adapted flies and to investigate the relative contributions of genetics versus environment to these differences.
Project description:Low oxygen conditions are not only common to natural environments but also occur during tissue invasive growth in the human host. Only few cellular factors have been identified up to now that allow the fungus to efficiently adapt its energy metabolism to the lack of O2. In the present study, we cultivated A. fumigatus in an O2-controlled fermenter and analysed its minute-scale responses to O2 limitation. Transcriptome sequencing revealed a group of genes underlying a rapid and highly dynamic regulation. As an initial experimental setup, A. fumigatus was cultivated in an O2-controlled fermenter, which allowed minute-scale variations in O2-fluxes largely independent of other secondary effects. Cultures were initially grown at saturated O2 concentrations (100% saturation ≈ 260 µmol l-1) and rapidly shifted to hypoxic growth conditions at an initial O2 saturation of 5% (≈ 13 µmol l-1). Dynamics of low and high O2 responses of A. fumigatus cultivated in an O2-controlled fermenter
Project description:To delineate the role of hypoxia in esophageal epithelial biology, we carried out gene array experiments using a non-transformed immortalized diploid human esophageal cell line, EPC2-hTERT (Mol Cancer Res. 2003;1:729-38). Unlike cancer cell lines, EPC2-hTERT has no genetic alterations at early passages that may affect the cellular response to hypoxia. Experiment Overall Design: EPC2-hTERT cells were exposed to moderate (1% O2) hypoxia in experiment 1 (Exp1) or severe (0.2% O2) hypoxia in experiment 2 (Exp2). Normoxia (21% O2) served as a control in both experiments.
Project description:To realize the full potential of human embryonic stem cells (hESC), it is important to develop culture conditions that maintain hESC in a pluripotent, undifferentiated state. A low O2 atmosphere (~4% O2), for example, prevents spontaneous differentiation and supports self-renewal of hESC. To identify genes whose expression is sensitive to O2 conditions, microarray analysis was performed on RNA from hESC that had been maintained under either 4% or 20% O2. Of 149 genes differentially expressed, 42 were up-regulated and 107 down-regulated under 20% O2. Several of the down-regulated genes are most likely under the control of hypoxia-inducing factors and include genes encoding enzymes involved in carbohydrate catabolism and cellular redox state. Although genes associated with pluripotency, including OCT4, SOX2 and NANOG were generally unaffected, some genes controlled by these transcription factors, including LEFTY2, showed lowered expression under 20% O2, while a few genes implicated in lineage specification were up-regulated. Although the differences between O2 conditions were generally subtle, they were observed in two different hESC lines and at different passage numbers. The data are consistent with the hypothesis that 4% O2 favors the molecular mechanisms required for the maintenance of pluripotency. This report emphasizes the importance of employing physiological concentrations of O2 when culturing hESC. The transcript profiles of cultures under 20 % O2 suggest that the cells are more poised to differentiate than when they are under the lower 4 % O2 conditions and that the down-regulation of LEFTY2 under 20 % O2 may destabilize the network of genes maintaining ESC pluripotency. Finally, the association of HIF2A with undifferentiated but not differentiating cells is consistent with a particular role for that transcription factor in control of pluripotency. Keywords: different oxygen concentrations in hESC culture condition Overall design: The H1 and H9 hESC lines had been maintained in a physiological O2 (3-5% O2) atmosphere since passage 26 and 23, respectively. The both H1 and H9 cells were propagated under 4% O2 for seven passages on MEF feeder layer, thereafter the both cells were switched to Matrigel coated culture wells for at least three passages prior to RNA collection. RNA extraction was performed within 3 min following removal of the culture plates from respective O2 conditions and well before dissolved O2 in the culture medium showed any detectable changes. Total six RNA samples were collected from H1 cells at two different passage numbers (H1p37 and H1p50) and H9 cells at passage 32 (H9p32) under 4% and 20% O2 conditions, respectively. The cells maintained under 4% O2 were split into both 4% and 20% O2 conditions and cultured for 7 days before of RNA collection.