Project description:Pluripotent stem cells can be induced from somatic cells, providing an unlimited cell resource for regenerative medicine. However, genetic manipulation and difficult-to-manufacture strategies used in reprogramming limit their clinical applications. Here, we show pluripotency can be induced from mouse somatic cells by specific small-molecule compounds. The completely chemically-induced pluripotent stem cells (CiPSCs) can be stably maintained in embryonic stem cell (ESC) culture medium and resemble ESCs in terms of their gene expression profiles, epigenetic status, and potential for differentiation and germline transmission. These findings suggest that exogenous master genes are dispensable for cell fate reprogramming and pave the way for the clinical application of somatic reprogramming techniques. Pluripotent stem cells can be induced from somatic cells, providing an unlimited cell resource for regenerative medicine. However, genetic manipulation and difficult-to-manufacture strategies used in reprogramming limit their clinical applications. Here, we show pluripotency can be induced from mouse somatic cells by specific small-molecule compounds. The completely chemically-induced pluripotent stem cells (CiPSCs) can be stably maintained in embryonic stem cell (ESC) culture medium and resemble ESCs in terms of their gene expression profiles, epigenetic status, and potential for differentiation and germline transmission. These findings suggest that exogenous master genes are dispensable for cell fate reprogramming and pave the way for the clinical application of somatic reprogramming techniques. Chemicals' acronyms: V, VPA; C, CHIR; 6, 616452; T, tranylcypromine; F, FSK; Z, DZNep; P, PGE2; R, RG108; S, SRT1720; M, 2-Me-5HT; D, D4476; B, Sodium butyrate. To compare the global gene expression pofile of mouse embryonic fibroblasts (MEFs), mouse adult lung fibroblasts (MAFs), mouse neonatal fibroblasts (MNFs), adipose-derived stem cells (ADSCs), chemical induced pluripotent stem cells (CiPS), embryonic stem cells (ESCs) and OSKM-iPSCs. We profiled the mRNA expression of each sample by microarray (Mouse OneArray® v2, Phalanx). Different sample set was normalized for different purpose. Set1: MEFs1, CiPS34, MNF CIPS7, CiPS50 CiPS21, OSKM-iPS1 and ESCs1 were analyzed, each sample have three replicates; Set2: MNFs, MAFs, ADSCs, MNF CiPS1, MAF CiPS3, CiPS45, ADSC CiPS2, OSKM iPS2 and ESCs2 were analyzed, each sample have three replicates; Set3: WT MEFs, ESCs3, CiPS WT1 and CiPS WT2 were analyzed, each sample have two replicates; Set4: MEFs2, SKM-FSK1, SKM-FSK2 and ESCs4 were analyzed, each sample have three replicates; Set5: MEFs3, ESCs5, CiPSCs, D12, D20 and D32 were analyzed, each sample have two replicates.
Project description:To investigate the mechanisms by which C/EBPa drives basophil differentiation and maintains basophil identity, we examined whether or not C/EBPa promotes basophil molecular programming and simultaneously represses mast cell molecular programming. We performed genome-wide gene expression profiling on basophils and mast cells and found that 6798 genes were shared by mast cells and basophils; 2033 genes were expressed 2-10 (log2 1-3.3)-fold higher in basophils (differentially expressed in basophils); and 413 genes were expressed greater than 10 (log2 3.3)-fold in basophils (highly expressed in basophils). On the other hand, we found 569 genes were expressed 2-10 (log2 -1 to -3.3) fold higher in mast cells and 171 genes were highly expressed in mast cells [greater than 10 fold (log2 -3.3)]. We treated purified basophils prepared from Cebpaf/f RosaYFP/creER mice and Cebpa+/+ RosaYFP/creER control mice with or without 4HT treatment for five days. Gene expression in the treated basophils was analyzed using microarray analysis. Overall, deletion of C/EBPa in basophils resulted in a reduction of mRNA expression for 248 genes and led to an increase in mRNA expression for 255 genes. The majority of the C/EBPa-regulated genes were either differentially or highly expressed in basophils or mast cells. In this study, we compared gene expression in basophils and mast cell and identified genes which specifically expressed in basophils and mast cells. By using Cebpa conditional knock out mice, we identified Cebpa regulated genes in basophils.
Project description:The serine/threonine protein kinase paralogs ROCK1 & 2 have been implicated as essential modulators of angiogenesis; however their paralog-specific roles in endothelial function are unknown. Microarray analysis of ROCK knockdown lines revealed overlapping and unique control of global transcription by the paralogs, and a reduction in the transcriptional regulation of just under 50% of VEGF responsive genes. MS1 mouse endothelial cells were stably transfected with non-targeting, ROCK1 specific, or ROCK2 specific shRNA plasmids. Cells were grown to confluence and treated with sham or 2.5 ng/ml human recombinant vascular endothelial growth factor (VEGF) for 12 hours. RNA from six independent biological replicates from each engineered cell line were collected, pooled, and subjected to triplicate technical replicates of microarray analysis.
Project description:Autophagy to apoptosis switching event is an unexplored area. We were interested to explore the gene expression profile at this juncture. Our Microarray data emphasized that among all eNOS and p62 genes were markedly induced. In fuctional level eNOS expression activates mTORC1 followed by inhibition of autophagy. This ultimately allowed accumulation of p62 . Intraccellular accumulation of p62 sequestered unfolded toxic protein aggregates in mitochondria and ER resulting in to impaired redox regulation. Intraccelular ROS generation further sensitized cells for apoptosis and tilted autophagic response to apoptosis. Cells were treated with Tunicamycin as an inducer of both autophagy and apoptosis. At early stage of Tunicamycin treatment 24h , prostate cancer cell PC3 showed activation of autophagic process where apoptosis was not evident. Sustained ER stress with Tunicamycin induced late apoptoisis at 72h of treatment. Hence we isolated total RNA from Untreated cells, cells with Tunicamycin treatment after 24h and 72h. Further the RNA were used to perform whole genome microarray to analyze the gene expression profile at autophagy and apoptosis juncture. The selected genes were also validated by qPCR and western bot. Morover RNAi study were implemented to evaluate the signaling crosstalk at the juncture of autophagy and apoptosis.
Project description:The current study set to determine the effects of Fibroblast Growth Factor 2 and cell culture surface (tissue culture plastic and glass) on the transcriptome of adult human dermal fibroblasts. Transcriptional profiles of adult human dermal fibroblasts grown in four experimental conditions (glass, glass and addition of FGF2, plastic, plastic and addition of FGF2) were compared. Comparison of the transcriptomes will allow to identify significantly differentially expressed genes due to FGF2 and surface treatment, which in turn will allow for identification of the pathways affected by these factors in the human adult fibroblasts. Transcriptome analysis of adult human dermal fibroblasts grown on tissue culture plastic and glass, with and without 4ng/ml FGF2, was performed in two biological replicates and two technical replicates for each treatment condition.
Project description:We studied the radiation effect on cell culture model of oral mucositis. The musositis model (tissues) used is an organotypic model which consisted of 3-dimensional (3-D) cell cultures of primary human oral keratinocytes. The tissues were purchased from MatTek corporation (Ashland, MA) and were grown on top of microporous membrane and supplemented with MatTek serum free media. These tissues were irradiated with 12 Gy. Six hours after the irradiation the tissues were cut in half. Half of the tissue was used to extract total RNA (RNeasy Plus Mini kit, Qiagen, Germantown, MD) and the other half was used to evaluate the histology and the apoptosis. We had the following groups: Non-Irradiated Tissue, Irradiated Tissue with 12 Gy; Irradiated Tissue with 12 Gy pretreated with N-acetylcysteine (ACS); Irradiated Tissue with 12 Gy pretreated with the Traditional Chinese Medicine (TCM) Qingre-Liyan decoction; and Irradiated Tissue with 12 Gy pretreated with 50:50 w/w ACS:TCM (AT) Group 1: Human Prirmary 3-D Cultures of Human Keratinocytes were irradiated with 12 Gy; Group 2: Human Prirmary 3-D Cultures of Human Keratinocytes were pretereated with N-acetylcysteine (ACS) and then irradiated with 12 Gy; Human Prirmary 3-D Cultures of Human Keratinocytes were pretreated with the Traditional Chinese Medicine (TCM), Qingre-Liyan Decostion and then irradiated with 12 Gy; Human Prirmary 3-D Cultures of Human Keratinocytes were pretreated with a 50:50 w/w mixture of ACS:TCM (AT) and then irradiated with 12 Gy. Each sample was done in triplicate.
Project description:Baseline gene expression of mouse Cardiac Progenitor Cells (CPC) We used microarrays to analyze the global gene expression of mouse CPCs. Compare the gloable gene expression of mouse CPCs and Cardiomyocytes. Although the chip is two color, we only used Cy5 as the data channel.
Project description:Elaidic acid is an abundant industrial produced trans fatty acid in foodstuffs. Trans fatty acid intake has been correlated to an unfavorable plasma lipoprotein profile and an increased cardiovascular disease risk. The present study aimed to identify a plasma protein biomarker panel related to human intake of elaidic acid. The human liver cell line HepG2-SF was used as a model system, and the cells were maintained for seven days in serum-free medium containing 100 µM elaidic acid (trans∆9-C18:1), oleic acid (cis∆9-C18:1) or stearic acid (C18:0). The secretomes were analyzed by stable isotope labeling of amino acids in cell culture (SILAC), difference in gel electrophoresis (DIGE) and gene expression microarray analysis. Twelve proteins were found to be differentially regulated based on SILAC data (>1.3 fold change, P-value < 0.05), 13 proteins were found to be differentially regulated based on DIGE analysis (>1.3 fold change, P-value < 0.05), and 17 mRNA transcripts encoding extracellular proteins were determined to be affected (>1.3 fold change, P-value < 0.01) following the addition of elaidic acid compared to oleic acid or stearic acid. The results revealed that 37 proteins were regulated specifically in response to elaidic acid exposure, and nine of these proteins were confirmed to be regulated in this manner by using selected reaction monitoring mass spectrometry. HepG2 cells kept used in serum free conditions. The cells were incubated for seven days in either 100 µM oleic, elaidic or stearic acid, before total RNA was extracted. For each group four biological replicates were made and from the each RNA extraction two technical replicates were made. There is a control group without fatty acid incubation.
Project description:A human cDNA microarray comprising 29,187 human genome probes was used to evaluate the transcriptional changes between brian glioma stem cells (GSC) and normal neural stem cells(NSC). There were 6 samples that were analyzed, 3 samples were from glioma stem cells and the other 3 samples form normal neural stem cells were controls. cell type: brain glioma stem cells:GSC_H004, GSC_H005, GSC_H006 ; cell line: normal neural stem cells: NSC_H001, NSC_H002, NSC_H003 biological replicate: NSC_H001, NSC_H002, NSC_H003; biological replicate: GSC_H004, GSC_H005, GSC_H006