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Effects of Essential 8 (E8) media additives polyethylene glycol (PEG) and Heparin sodium salt (HS) on the pluripotency and oncogenes

ABSTRACT: Manuscript Name: "Addressing Bioreactor hiPSC Aggregate Stability, Maintenance and Scaleup Challenges Using a Design of Experiment Approach" Evaluation of core pluripotent genes showed that expression levels were similar between samples with a clear indication of a primed phenotype. In addition, we evaluated several well-known oncogenes notable TP53, MYC, NANOGP8, EEF1A2 and KLF4 were expressed with similar transcript levels between samples, though KLF4 had low overall expression. It was observed that a few genes indicative of forward differentiation were expressed with TSR genes TDGF1, LEFTY1 and IL17RC and the trophectodermal genes KRT8 and TEAD4 showing comparable expression levels. Expression of the primary primitive streak gene NODAL was observed at low transcript levels in both samples. Examining the top differentially expressed genes we noted that there were several genes involved in ectodermal differentiation and some key signaling pathway components that were significantly downregulated in response to the presence of PEG and HS. Down-regulated ectodermal genes included SOX1, OLIG3, LHX5 and OTX2 as well as some less known ones. The pathways most downregulated were the TGFβ family as suggested by BMP2 and BMP4 down regulation, though the previously mentioned levels of TDGF1, LEFTY1 and NODAL remained consistent to cultures lacking additives. Wnt family signaling components down regulated were Wnt4, FRZB, FZD5 and FRZ8. Also a decreased in retinoic acid signaling components were noted as indicated by decreased CYP26A1, CYP26C1, DHRS3 and CRABP2 transcript levels.

ORGANISM(S): Homo sapiens

PROVIDER: GSE264081 | GEO | 2024/04/16

REPOSITORIES: GEO

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Project description:Mammalian genomes are subjected to epigenetic modifications, including cytosine methylation by DNA methyltransferases (Dnmt) and further oxidation by Ten-eleven-translocation (Tet) family of dioxygenases. Cytosine methylation plays key roles in multiple processes such as genomic imprinting and X-chromosome inactivation. However, the functional significance of cytosine methylation and the further oxidation has remained undetermined in mouse embryogenesis. Here we show that global inactivation of all three Tet genes in mice led to consistent defects in gastrulation. The defects include reduced specification of the axial mesoderm and paraxial mesoderm, mimicking phenotypes in embryos with gain-of-function Nodal signaling, a cardinal cue for gastrulation. Introduction of a single mutant allele of Nodal in the Tet mutant background partially restored patterning, suggesting that hyperactive Nodal signaling is a leading cause for the gastrulation failure of Tet mutants. Increased Nodal signaling is likely due to diminished expression of the Lefty1 and Lefty2 genes, inhibitors of Nodal signaling. Moreover, reduction in the Lefty gene expression can be ascribed to elevated DNA methylation as both Lefty-Nodal signaling and normal morphogenesis are largely restored in Tet-deficient embryos when the Dnmt3a and Dnmt3b genes are disrupted. Additionally, specific inactivation of Tet by point mutations abolishing the dioxygenase activity causes similar molecular and gastrulation abnormalities. Taken together, our results show that Tet-mediated DNA oxidation modulates the Lefty-Nodal signaling by promoting demethylation of the shared target genes with Dnmt3a and Dnmt3b. These findings reveal a fundamental epigenetic mechanism featuring dynamic DNA methylation and demethylation and their role in the regulation of key signaling in body plan formation during early embryogenesis. Examine RNA expression and DNA methylation differences between Tet-null and wild type samples of mouse epiblast in E6.5.

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Effects of Essential 8 (E8) media additives polyethylene glycol (PEG) and Heparin sodium salt (HS) on the pluripotency and oncogenes

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