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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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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. 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

ORGANISM(S): Mus musculus

SUBMITTER: Takaho Endo

PROVIDER: E-GEOD-70307 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

2016-06-26 | GSE70307 | GEO

Mus musculus

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

| PRJNA288290 | ENA

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

Role of de novo DNA methyltransferases in hematopoietic stem cells (RNA-seq)

Project description:To investigate the global transcriptome expression in Dnmt3ab dKO HSCs, we performed high-throughput sequencing of Poly A+ RNA (RNA-Seq) from purified HSCs (SP-KSL-CD150+). With biological duplicates, more than 200 million reads in total for each genotype of HSC were obtained. Mouse hematopoietic stem cell mRNA profiles of Dnmt3ab Dko HSCs, generated by deep sequencing, in duplicate, using Illumina Hiseq 2000

2014-08-19 | E-GEOD-50775 | biostudies-arrayexpress

The induction of integrin beta3 signaling in hematopoietic stem cells (HSCs)

Project description:To determine the molecules involved in beta3 integrin signaling in HSCs, we carried out DNA microarray analyses using HSCs stimulated with ligand-mimic antibody for intetegrin beta3 in the presence of stem cell factor(SCF) and/or thrombopoietin (TPO). All experiments were performed in duplicate or triplicate using sorted cells that were independently prepared in separate experiments. After CD34-KSL HSCs were cultured with 2C9.G2 or control IgG for 5 days in S-Clone SF-03 medium supplemented with 50 ng/ml SCF and/or 50 ng/ml TPO, gene expression profiles were examimed using 5,000 sorted CD48-KSL cells from each sample.

2011-11-16 | E-GEOD-33696 | biostudies-arrayexpress

Epigenetic and genetic changes during mouse hematopoietic stem cell aging [RNA-Seq]

Project description:To investigate the global transcriptome changes in mouse hematopoietic stem cell aging, we performed high-throughput sequencing of Poly A+ RNA (RNA-Seq) from purified 4 month, and 24 month-old HSCs (SP-KSL-CD150+). With biological duplicates, more than 200 million reads in total for each age of HSC were obtained. Comparison of the young and old HSC transcriptomes revealed that 1,337 genes that were up-regulated, and 1,297 genes were down-regulated with HSC aging. The most highly represented upstream regulator was growth factor TGFB1, accounting for ~ 19% of differential gene expression in young versus old HSC (p-value = 1.96E-33). Gene ontology (GO) analyses indicated that up-regulated genes in 24mo HSCs are highly enriched in Regulation of Cell Adhesion, Regulation of Cell Proliferation and Ribosome, while down-regulated genes are enriched in DNA Base Excision Repair, DNA replication and Cell Cycle. RNA-seq also allowed us to examine alternative isoforms with aging including alternative splicing, promoter usage and pre-mRNA abundance. Mouse hematopoietic stem cell mRNA profiles of 4 month and 24 month old WT mice were generated generated by deep sequencing, in duplicate, using Illumina Hiseq 2000

2014-04-30 | E-GEOD-47817 | biostudies-arrayexpress

Expression data 550cGy vs 0cGy KSL

Project description:Precise study of HSCs during regeneration has been impeded by the rarity of the HSC population and depletion of phenotypic HSCs early following genotoxic stresses, such as total body irradiation (TBI). We isolated bone marrow (BM) ckit+sca-1+lin- (KSL) cells, which are enriched for HSCs, from adult C57Bl6 mice before and at several time points following TBI, as a means to map the dynamic molecular response of HSC regeneration. We isolated BM KSL cells and myeloid progenitor cells (c-kit+sca-1-lin- cells) at day +14 after irradiation and compared the gene expression profile of regenerating HSCs versus steady state HSCs (non-irradiated) and committed progenitor cells.

2016-04-05 | E-GEOD-79744 | biostudies-arrayexpress

Epigenetic and genetic changes during mouse hematopoietic stem cell aging [Bisulfite-Seq]

Project description:To investigate the global DNA methylation changes in mouse hematopoietic stem cell aging, we performed whole-genome bisulfite sequencing (WGBS). We generated 1,494 million (4mo HSCs), and 1,493 million (24mo HSCs) raw reads; about 82.6% and 84.3%, respectively, were successfully aligned to either strand of the reference genome (mm9). Of all the cytosines present in the reference genome sequence, about 93% of Cs and 99% of CGs were covered in both datasets, with an average coverage of 46-fold (4mo) and 50-fold (24mo). In contrast to the age-associated hypomethylation observed in studies of somatic cells, mentioned above, HSCs showed an increase of methylation with age. The average methylation level over all 16 million covered CpGs increased from 83.5% in young (4mo) HSC to 84.6% in old (24mo) HSC. We observed a total of 448,166 differentially methylated CpGs (DMCs), defined as those having a 20% or more difference in methylation ratio, of which 38.5% (172,609) were hypomethylated (hypo-DMCs) and 61.5% (275,557) were hypermethylated (hyper-DMCs) with aging. For different genomic features, a slightly greater DNA methylation ratio increase was observed for the gene body, LINEs and SINEs, while CGIs and promoters showed balanced increases and decreases. Localization analysis of DMCs indicates that DNA encoding for ribosome RNA (rDNA) is primarily a hotspot for hypo-DMCs, while promoters without CpG islands, CpG island shores and LINE repetitive elements exhibit both hypo- and hyper-DMCs. Mouse hematopoietic stem cell DNA methylation profiles of 4 month and 24 month old WT mice were generated generated by deep sequencing, in duplicate, using Illumina Hiseq 2000.

2014-04-30 | E-GEOD-47815 | 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

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