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

Purpose: The goals of this study are to elucidate the influence of integrin ß3 signaling on STAT1-dependnet gene expression in IFN?-treated HSCs. Methods: Wild type (WT) HSCs were cultured with or without IFN? 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 IFN?, mRNA profiles of STAT1-/- HSCs treated with or without IFN? 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 IFN?-treated STAT1-/- HSCs was categorized to the group including Wt HSCs cultured in the absence of IFN? rather than HSCs treated with IFN?. 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 IFN?. In contrast, integrin ß3 signaling in the absence of IFN? appears to not influence the expression of IFN?/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 IFN?-mediated HSC responses and integrin ß3 signaling in HSCs promotes STAT1-dependent gene expression in the presence of IFN?. 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).

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Umemoto T, Matsuzaki Y, Shiratsuchi Y, Hashimoto M, Yoshimoto T, Nakamura-Ishizu A, Petrich B, Yamato M, Suda T