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Global gene expression profiling experiments of HSC cultured in cysteine or valine deficient medium by RNA-sequencing analysis

ABSTRACT: We investigated the role of amino acid to maintain HSC function. To identify essential amino acids for HSCs, CD34-KSL cells were cultured in single amino acids deficient medium. And cultured cells were transplanted into lethally irradiated mice. Then, the donor chimerism and lineage contribution was estimated. Surprisingly, HSC proliferation was prevented in valine and cysteine deficient medium in vitro. Donor cells cultured in these medium were also not engrafted. To elucidate the effects and influences of cysteine and valine in HSCs, we performed global gene expression profiling experiments by RNA-sequencing analysis. Gene sets categorized with cell cycle, mitosis, cell division or DNA replication were strongly down-regulated in both valine- or cysteine-depleted conditions These results imply distinctive amino-acid metabolism involved in HSC division.

ORGANISM(S): Mus musculus

PROVIDER: GSE70307 | GEO | 2016/06/26

SECONDARY ACCESSION(S): PRJNA288290

REPOSITORIES: GEO

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global gene expression profiling experiments of HSC cultured in cysteine or valine deficient medium by RNA-sequencing analysis

Project description:We investigated the role of amino acid to maintain HSC function. To identify essential amino acids for HSCs, CD34-KSL cells were cultured in single amino acids deficient medium. And cultured cells were transplanted into lethally irradiated mice. Then, the donor chimerism and lineage contribution was estimated. Surprisingly, HSC proliferation was prevented in valine and cysteine deficient medium in vitro. Donor cells cultured in these medium were also not engrafted. To elucidate the effects and influences of cysteine and valine in HSCs, we performed global gene expression profiling experiments by RNA-sequencing analysis. Gene sets categorized with cell cycle, mitosis, cell division or DNA replication were strongly down-regulated in both valine- or cysteine-depleted conditions These results imply distinctive amino-acid metabolism involved in HSC division. Gene expression profiles of ten thousand HSCs cultured in cysteine or valine deficient medium for 24 hours were compared with that of HSCs cultured in complete medium by using RNA-sequencing analysis

2016-06-26 | E-GEOD-70307 | biostudies-arrayexpress

Cysteine deprivation in liver cell line

2007-12-13 | GSE9517 | GEO

Transcription profiling of human liver cell line after cysteine deprivation

Project description:First experiment: Cells were cultured in sulfur amino acid-free DMEM supplemented with 0.1 mM methionine + 0.1 mM cysteine (complete) or supplemented only with 0.1 mM methionine (cysteine-free). Cells were cultured in either medium for 42 h (Long + Cys; Long -Cys) or in cysteine-free medium for 36 h followed by 6 h in complete medium (Short +Cys); Second experiment: C3A/HepG2 cells were cultured in sulfur amino acid-free DMEM supplemented with 0.1 mM Met and 0.1 mM Cys (complete) or supplemented only with 0.1 mM Met (cysteine-devoid). Cells were cultured in complete medium for 42 h (Long +Cys) or in complete medium for 36 h followed by cysteine-devoid medium for 6 h (Short -Cys). Experiment Overall Design: First experiment: Three plates of cells were cultured under each condition. Cells were cultured in sulfur amino acid-free DMEM supplemented with 0.1 mM methionine + 0.1 mM cysteine (complete) or supplemented only with 0.1 mM methionine (cysteine-free). Cells were cultured in either medium for 42 h (Long + Cys; Long -Cys) or in cysteine-free medium for 36 h followed by 6 h in complete medium (Short +Cys). Experiment Overall Design: Second experiment: C3A/HepG2 cells were cultured in sulfur amino acid-free DMEM supplemented with 0.1 mM Met and 0.1 mM Cys (complete) or supplemented only with 0.1 mM Met (cysteine-devoid). Cells were cultured in complete medium for 42 h (Long +Cys) or in complete medium for 36 h followed by cysteine-devoid medium for 6 h (Short -Cys).

2008-06-16 | E-GEOD-9517 | biostudies-arrayexpress

Mus musculus

Project description:global gene expression profiling experiments of HSC cultured in cysteine or valine deficient medium by RNA-sequencing analysis

| PRJNA288290 | ENA

B. subtilis: temporal expression response to amino acid treatment

Project description:Microorganisms can restructure their transcriptional output to adapt to environmental conditions by sensing endogenous metabolite pool. In this study, an Agilent customized microarray representing approximately 4,106 genes was used to study temporal transcript profiles of Bacillus subtilis in response to valine, glutamate and glutamine pulses. Amino-acid-regulated genes were identified having significantly changed expression at one or more time points in response to pulses of valine, glutamate, and glutamine, respectively, and Val-, Glu and Gln-specific genes were further distinguished from them. Different amino acid treatments were compared in terms of both the global temporal profiles and the 5-minute quick regulations, and between-experiment differential genes were identified. The highlighted genes were analyzed based on diverse sources of gene functions using a variety of computational tools, including T-profiler analysis, hierarchical clustering and enrichment of functional categories. The results revealed the common and distinct modes of action of these three amino acids, and should help to elucidate the specific signaling mechanism of each amino acid as an effector. Three amino acids (Glutamate, Glutamine, and Valine) were adopted to perturb the culture of subtilis. Four time-points were investigated for each perturbation. There are two replicates for the first time-point of Valine-treatment experiment.

2010-05-05 | E-GEOD-17243 | biostudies-arrayexpress

Genome-wide analysis of gene expression after tyrosine or methionine/cysteine deprivation in HeLa cells

2012-07-24 | GSE35323 | GEO

Genome-wide analysis of gene expression after tyrosine or methionine/cysteine deprivation in HeLa cells

Project description:Limitation of essential amino acids, such as tyrosine or methionine/cysteine, causes upregulation of exogenous integrated transgene expression in mammalian cells. This phenomenon is mediated by histone acetylation and chromatin remodelling, since histone deacetylase (HDAC) inhibitors reproduce starvation-induced transgene upregulation and chromatin immunoprecipitation analysis of amino acid-deprived cells reveals significant changes in total core histones detectable at the CMV promoter. Expression profiling of HeLa cells starved for 5 days in medium without tyrosine or methionine/cysteine provides important information on the cellular response to amino acid deprivation and suggests the involvement of HDAC4 (class II HDAC) in transgene derepression during amino acid starvation. Total RNA obtained from HeLa and HeLaOA1myc cells (HeLa cells with a human ocular albinism type 1 (OA1)+myc tag transgene) subjected to 5 days of tyrosine or methionine/cysteine starvation compared to control cells. Twelve samples are analyzed: HeLa and HeLaOA1myc grown for 5 days in RPMI deprived of tyrosine (Y) vs. complete RPMI; technical duplicates of HeLa and HeLaOA1myc grown for 5 days in DMEM deprived of methionine/cysteine (MC) vs. complete DMEM.

2012-07-23 | E-GEOD-35323 | biostudies-arrayexpress

Molecular characterization of hematopoietic stem cells after in vitro amplification on biomimetic 3D PDMS cell culture scaffoldsMolecular characterization of hematopoietic stem cells after in vitro amplification on biomimetic 3D PDMS cell culture scaffolds

Project description:Hematopoietic stem cell transplantation (HSCT) is successfully applied since the late 1950s, however, its efficacy still needs to be improved. A promising strategy is to transplant high numbers of pluripotent hematopoietic stem cells (HSCs). Therefore, an advanced ex vivo culture system is needed that supports the proliferation and maintains the pluripotency of HSC to override possible limitations in cell numbers gained from donors. To model the natural HSC niche in vitro and thus, to amplify high numbers of undifferentiated HSCs, we used an optimized HSC cell culture medium in combination with artificial 3D bone marrow-like scaffolds made of polydimethylsiloxane (PDMS). After 14 days in vitro (DIV) cell culture, we performed transcriptome and proteome analysis of the whole cell populations. Ingenuity pathway analysis (IPA) indicated that our 3D PDMS cell culture scaffolds activated interleukin, SREBP, mTOR and FOXO signaling pathways as well as the HSC metabolism, which we confirmed by ELISA, Western blot and metabolic flux analysis. These molecular signaling pathways and HSC metabolism are well known to promote the expansion HSCs and are involved in their pluripotency maintenance. After selection and enrichment of immature CD34-positive/CD38-negative HSCs using FACS sorting, we could confirm our findings by another proteome analysis followed by IPA. Thus, we could show that our 3D bone marrow-like PDMS scaffolds activate key molecular signaling pathways to amplify the numbers of undifferentiated HSC efficiently ex vivo.

2021-11-10 | PXD025849 | Pride

SILAC-based Quantitative Proteomic Analysis of Secretome between Activated and Reverted Hepatic Stellate Cells

Project description:Activated hepatic stellate cell (HSC) is the main myofibroblast cell in the liver fibrosis(LF). An important characteristic of the recovery of LF is not only the apoptosis of activated HSCs but also reversal of myofibroblast-like phenotype to a quiescent-like phenotype. Understanding the changes of secreted proteins in the reversion of activated HSCs may provide the broader view of cellular regulatory networks and discover candidate markers or targets for therapeutic strategies of LF. In this study, stable isotope labeling with amino acids (SILAC) combining with LTQ-FT mass spectrometer were performed on in vitro activated HSCs and reverted HSCs to obtain a proteomic view of secretory proteins. In total, 330 proteins showed significant differences in reverted HSCs. Among these, 109 up-regulated proteins were mainly involved in amino acid metabolism pathway and glucose metabolism pathway using GeneGO/MetaCore software, while 221 down-regulated proteins are closely associated with HSCs activation, such as cytoskeleton remodeling, chemokines and cell adhesion. Additionally, a set of novel proteins associated with HSCs activation and reversion were validated by Western blotting in the cell secretion and in the sera of LF, including Vitronectin, laminin beta 1(LAMB1) and Ubiquitin conjugation factor E4B(UBE4B). Our study provided the valuable insight into the mechanisms in the reversion of activated HSCs and identified some potential biomarkers of LF in clinical studies.

2014-08-11 | PXD000773 | Pride

Gene expression at single cell level in human umbilical cord blood cells enriched for hematopoietic stem cells and cultured in conditions that maintain HSCs ex vivo.

Project description:The capacity for self-renewal of human hematopoietic stem cells (HSCs) is lost upon typical culture conditions, which often include high levels of multiple hematopoietic cytokines, serum, and/or support cells. We found that culture of HSCs in CR medium (basal culture medium with inhibitors of GSK-3 (CHIR99021) and mTORC1 (Rapamycin) and no serum or cytokines) maintains self-renewal in human HSCs for at least 7 days without loss of repopulating activity (Huang et al PMID 23142822). Here we performed single cell RNA-seq to determine transcriptomic changes associated with HSC maintenance in CR. We compared a population of freshly isolated human hematopoietic cells enriched for HSCs (CD34+CD38-CD45RA-CD90+) to cells cultured for 2 days in basal medium (vehicle control), CR, and CR and low dose cytokines (CRCY). The data show that, compared to Day 0 uncultured cells, the HSC pool is maintained when CR is included (CR or CRCY medium) but not in the vehicle control. The most highly enriched transcript in the HSC population was the chaperone/UPR sensor GRP78/HSPA5. This population was also highly enriched for immediate early response genes and components of the AP-1 complex (FOS and JUN family transcription factors) which mediate the immediate early response to stress.

2023-12-08 | GSE248311 | GEO

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