Project description:Analysis of genes that were differentially expressed in mutant VUB03_DM1 as compared to controls VUB01 and SA01 Mesodermal Precursors Cells. Embryonic stem (ES) cell lines provide, theoretically, unlimited access to any needed amount of any specific cell phenotype of an organism, due to their unique capacities at indefinite self-renewal and pluripotency (Smith 2001; Trounson 2006). These properties allow using the progeny of ES cell lines to model human pathologies (Martinat, Shendelman et al. 2004; Lerou and Daley 2005; Ben-Nun and Benvenisty 2006). In particular, human ES cell lines derived from embryos characterized as gene-carriers following pre-implantation genetic diagnosis (PGD) for any one of major monogenic diseases (Pickering, Minger et al. 2005; Mateizel, De Temmerman et al. 2006) may be considered as perfect cellular replicas of those diseases, as they exhibit the exact genotypes associated to them. Here, we confirm this hypothesis by demonstrating that the cell progeny of an ES cell line derived from an embryo with myotonic dystrophy type 1 (DM1) displayed the morphological stigma associated to the expression of the mutant gene âso-called intranuclear foci- as well as abnormal alternate splicing of the insulin receptor, a characteristic feature of DM1. Further differential transcriptomic analysis of the DM1 gene-carrying cells with phenotypically similar populations from native ES cell lines revealed abnormal expression of 89 genes, among which 48 were down-regulated and 39 over-expressed. This study demonstrates that DM1, though a disease with relatively late clinical onset, is associated with expression of genetic defects early on during development. It underlines the value of PGD-derived ES cell lines as a tool to decipher molecular mechanisms of genetic diseases. Ben-Nun, I. F. and N. Benvenisty (2006). Human embryonic stem cells as a cellular model for human disorders. Mol Cell Endocrinol 252(1-2): 154-9. Lerou, P. H. and G. Q. Daley (2005). Therapeutic potential of embryonic stem cells. Blood Rev 19(6): 321-31. Martinat, C., S. Shendelman, et al. (2004). Sensitivity to oxidative stress in DJ-1-deficient dopamine neurons: an ES- derived cell model of primary Parkinsonism. PLoS Biol 2(11): e327. Mateizel, I., N. De Temmerman, et al. (2006). Derivation of human embryonic stem cell lines from embryos obtained after IVF and after PGD for monogenic disorders. Hum Reprod 21(2): 503-11. Pickering, S. J., S. L. Minger, et al. (2005). Generation of a human embryonic stem cell line encoding the cystic fibrosis mutation deltaF508, using preimplantation genetic diagnosis. Reprod Biomed Online 10(3): 390-7. Smith, A. G. (2001). Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol 17: 435-62. Trounson, A. (2006). The production and directed differentiation of human embryonic stem cells. Endocr Rev 27(2): 208-19. Keywords: disease state analysis Two controls hES-derived MPC (VUB01 and SA01) and one mutant hES-derived MPC (VUB03_DM1), with three biological replicats for each

Project description:Purpose: Liver-specific MPC-knockout (Mpc1-/-, LivKO) mice develop less liver tumors than wild-type (Mpc1+/+) mice when given a DEN/CCl4 hepatocarcinogenesis protocol. The goals of this study are to compare transcriptome changes (RNA-seq) between liver tumor and normal-adjacent tissue in WT and Mpc1-/- mice. Methods: Total RNA was collected from tumor and paired normal-adjacent liver samples using the Qiagen miRNeasy kit. RNA from four samples each of wild-type tumor (WT-Tumor), paired wild-type normal-adjacent (WT normal adjacent), Mpc1-/- (MPC LivKO) tumor (MPC LivKO-Tumor), and paired MPC LivKO normal-adjacent (MPC LivKO normal adjacent) tissue was isolated. Each tumor and its paired normal-adjacent tissue were analyzed in a paired manner. Library preparation and sequencing were performed using the Illumina mRNA-Seq workflow. For data normalization, the raw number of reads for each transcript was converted to Fragments Per Kilobase of transcript per Million mapped reads (FPKM). FPKM values were log transformed, and unsupervised clustering was performed on samples based on normalized expression of genes with variation in Euclidean distance among samples of at least 2.5 standard deviations using Cluster 3 software. Results: Using an optimized data analysis workflow, we mapped about 50 million sequence reads per sample to the mouse genome (buildmm10) and identified 15,777 transcripts in the liver tissue samples of WT an Mpc1-/- (MPC LivKO) with Illumina workflow. FPKM values were log transformed, and unsupervised clustering was performed using Cluster 3 software. Unsupervised clustering analysis identified six gene expression groups: (1) increased gene expression in both WT and LivKO tumors, (2) increased gene expression in WT tumors, (3) increased gene expression in MPC LivKO tumors, (4) decreased gene expression down in both WT and LivKO tumors, (5) decreased gene expression in WT tumors, and (6) decreased gene expression in MPC LivKO tumors. Conclusions: Our study is the first on Mpc1-/- liver tumors. The HCC markers Alpha-fetoprotein (Afp) and Glypican-3 (Gpc3) were in the cluster of genes upregulated in both WT and MPC LivKO tumors. In the cluster of 14 genes up-regulated in only WT tumors were two GSTs: Gsta1 and Gstp2. In the cluster of 108 genes down-regulated in only MPC LivKO tumors were three GSTs: Gsta2, Gsta3, and Mgst1. That same cluster contained Gpx1, glutathione peroxidase (Gpx1). Thus, we concluded WT tumors increased but MPC LivKO tumors decreased expression of glutathione metabolizing genes.

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