| Popis: |
Embryonic stem cells are derived from the inner cell mass of a blastocyst-stage embryo and are characterized by the ability to self-renew and differentiate into all cell types of an adult organism, as demonstrated by their transplantation into embryos in the mouse. Isolation of cells with similar properties from human embryos has permitted the study of human cell differentiation in vitro as might occur during development. As such, human ES cells may be useful to assess and predict the developmental toxicity of environmental compounds capable of epigenetic alterations of the genome and its expression. The first objective of my research was to validate the functional significance to maintenance of an undifferentiated human ES cell state of expressed genes whose epigenetic modification is conserved across diverse lines and/or likely to be deterministic of an embryo stem cell associated epigenetic state. The second goal was to determine the sensitivity and relationship of the expression of these genes to environmental factors known to perturb the epigenome, specifically subcytotoxic exposure to diverse organic and metallic compounds and the availability of atmospheric oxygen. siRNA-mediated knockdown of genes previously identified on the basis of the conserved methylation status of gene associated Cytosine-Guanine islands (i.e. GLIS2, HMGA1, PFDN5, TET1 and JMJD2C) and two related family members (TET2 & 3) resulted in induction of cell differentiation in two independent human ES cell lines (RH1 and H9). Differentiation was reflected by morphological changes, reduction or loss of pluripotency associated markers, qualitative and quantitative reduction in genomic 5-hmC and upregulation of diverse germinal lineage markers. Subcytotoxic exposure of the same human ES cell lines to diverse compounds known to alter the epigenome (i.e. 5-azacytidine, sodium arsenite, cadmium chloride and valproic acid) generally induced downregulation of the aforementioned genes, loss of genomic hydroxymethylation and differentiation when applied under normoxia (20% O2), the exception being valproic acid. The same treatment applied under hypoxia (0.5% O2), did not induce differentiation, with the exception of cadmium chloride. Hypoxia is a general feature of developing embryos prior to the establishment of a maternal/fetal placental interface and fetal cardiovasculature. The protective effect of hypoxia was associated with elevation of ROS, expression of the dioxygenases TET1 and JMJD2C, and genomic hydroxymethylation. This research has demonstrated that genes identified on the basis of a conserved pattern of epigenetic modification function in the maintenance of an undifferentiated human ES cell phenotype. Furthermore, a human ES cell-based toxicology test system has been developed with which one can assess the subcytotoxic effects of compounds known to disrupt the epigenome and affect development by assessing their impact on maintenance of an undifferentiated human ES cell state. This is reflected by alterations in pluripotency markers, epigenetically-defined biomarkers and changes in global 5-hmC levels and the expression of genes responsible for this epigenetic modification (TET1-3). The epigenetically-defined biomarkers of pluripotent human ES cell identity (GLIS2, HMGA1, PFDN5, JMJD2C and TET1) could serve as biomarkers for screenings of compounds at an epigenetic level as their expression has been shown to be altered upon compound exposure along with monitoring the expression of 5-hmC. |
