mRNA profiles of hematopoieitc stme cells treated with interferon gamma and/or vitronectin. Mus musculus

Purpose: The goals of this study are to elucidate the influence of integrin β3 signaling on STAT1-dependnet gene expression in IFNγ-treated HSCs. Methods: Wild type (WT) HSCs were cultured with or without IFNγ and/or VN in the presence of stem cell factor (SCF) plus thrombopoietin (TPO). Subsequently, cultured HSC fraction (CD48- c-kit+ Sca-1+ Lineage-) were sorted, followed by mRNA sequence using Ion Proton (n>4). Moreover, to extract genes whose expression were changed via STAT1 in the presence of IFNγ, mRNA profiles of STAT1-/- HSCs treated with or without IFNγ were also generated by the same way. The sequence reads that passed quality filters were analyzed by CLC genomic workbench. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm10) with CLC genomic workbench. Indeed, hierarchical clustering analysis showed that IFNγ-treated STAT1-/- HSCs was categorized to the group including Wt HSCs cultured in the absence of IFNγ rather than HSCs treated with IFNγ. Furthermore, gene set enrichment analysis (GSEA) showed that STAT1-dependent upregulated gene sets were significantly enriched within genes whose expression was enhanced in HSCs treated with VN and IFNγ. In contrast, integrin β3 signaling in the absence of IFNγ appears to not influence the expression of IFNγ/STAT1-dependent genes, as evidenced by the observation that VN treatment was statistically and significantly independent of the enrichment of gene sets that were both up-regulated by STAT1 Conclusions: Our study represents that STAT1 plays a central role in IFNγ-mediated HSC responses and integrin β3 signaling in HSCs promotes STAT1-dependent gene expression in the presence of IFNγ. Overall design: After HSCs derived from wild type (WT) and STAT1-/- mice were treated with IFNg and/or vitronectin for 5 days, mRNA profiles were generated by deep sequencing using Ion Proton system (n>4).