Low-dose radiation induces unstable gene expression in developing human iPSC-derived retinal ganglion organoids.

The effects of low-dose radiation on undifferentiated cells carry important implications. However, the effects on developing retinal cells remain unclear. Here, we analyzed the gene expression characteristics of neuronal organoids containing immature human retinal cells under low-dose radiation and predicted their changes. Developing retinal cells generated from human induced pluripotent stem cells (iPSCs) were irradiated with either 30 or 180 mGy on days 4-5 of development for 24 h. Genome-wide gene expression was observed until day 35. A knowledge-based pathway analysis algorithm revealed fluctuations in Rho signaling and many other pathways. After a month, the levels of an essential transcription factor of eye development, the proportion of paired box 6 (PAX6)-positive cells, and the proportion of retinal ganglion cell (RGC)-specific transcription factor POU class 4 homeobox 2 (POU4F2)-positive cells increased with 30 mGy of irradiation. In contrast, they decreased after 180 mGy of irradiation. Activation of the "development of neurons" pathway after 180 mGy indicated the dedifferentiation and development of other neural cells. Fluctuating effects after low-dose radiation exposure suggest that developing retinal cells employ hormesis and dedifferentiation mechanisms in response to stress.

iGEM as a human iPS cell-based global epigenetic modulation detection assay provides throughput characterization of chemicals affecting DNA methylation.

Chemical-induced dysregulation of DNA methylation during the fetal period is known to contribute to developmental disorders or increase the risk of certain diseases later in life. In this study, we developed an iGEM (iPS cell-based global epigenetic modulation) detection assay using human induced pluripotent stem (hiPS) cells that express a fluorescently labeled methyl-CpG-binding domain (MBD), which enables a high-throughput screening of epigenetic teratogens/mutagens. 135 chemicals with known cardiotoxicity and carcinogenicity were categorized according to the MBD signal intensity, which reflects the degree of nuclear spatial distribution/concentration of DNA methylation. Further biological characterization through machine-learning analysis that integrated genome-wide DNA methylation, gene expression profiling, and knowledge-based pathway analysis revealed that chemicals with hyperactive MBD signals strongly associated their effects on DNA methylation and expression of genes involved in cell cycle and development. These results demonstrated that our MBD-based integrated analytical system is a powerful framework for detecting epigenetic compounds and providing mechanism insights of pharmaceutical development for sustainable human health.

Epigenetic effects of insecticides on early differentiation of mouse embryonic stem cells.

Increasing evidence indicates that many insecticides produce significant epigenetic changes during embryogenesis, leading to developmental toxicities. However, the effects of insecticides on DNA methylation status during early development have not been well studied. We developed a novel nuclear phenotypic approach using mouse embryonic stem cells harboring enhanced green fluorescent protein fused with methyl CpG-binding protein to evaluate global DNA methylation changes via high-content imaging analysis. Exposure to imidacloprid, carbaryl, and o,p'-DDT increased the fluorescent intensity of granules in the nuclei, indicating global DNA methylating effects. However, DNA methylation profiling in cell-cycle-related genes, such as Cdkn2a, Dapk1, Cdh1, Mlh1, Timp3, and Rarb, decreased in imidacloprid treatments, suggesting the potential influence of DNA methylation patterns on cell differentiation. We developed a rapid method for evaluating global DNA methylation and used this approach to show that insecticides pose risks of developmental toxicity through DNA methylation.

Early Transcriptomic Changes upon Thalidomide Exposure Influence the Later Neuronal Development in Human Embryonic Stem Cell-Derived Spheres.

Stress in early life has been linked with the development of late-life neurological disorders. Early developmental age is potentially sensitive to several environmental chemicals such as alcohol, drugs, food contaminants, or air pollutants. The recent advances using three-dimensional neural sphere cultures derived from pluripotent stem cells have provided insights into the etiology of neurological diseases and new therapeutic strategies for assessing chemical safety. In this study, we investigated the neurodevelopmental effects of exposure to thalidomide (TMD); 2,2',4,4'-tetrabromodiphenyl ether; bisphenol A; and 4-hydroxy-2,2',3,4',5,5',6-heptachlorobiphenyl using a human embryonic stem cell (hESC)-derived sphere model. We exposed each chemical to the spheres and conducted a combinational analysis of global gene expression profiling using microarray at the early stage and morphological examination of neural differentiation at the later stage to understand the molecular events underlying the development of hESC-derived spheres. Among the four chemicals, TMD exposure especially influenced the differentiation of spheres into neuronal cells. Transcriptomic analysis and functional annotation identified specific genes that are TMD-induced and associated with ERK and synaptic signaling pathways. Computational network analysis predicted that TMD induced the expression of DNA-binding protein inhibitor ID2, which plays an important role in neuronal development. These findings provide direct evidence that early transcriptomic changes during differentiation of hESCs upon exposure to TMD influence neuronal development in the later stages.

Effects of Polyamidoamine Dendrimers on a 3-D Neurosphere System Using Human Neural Progenitor Cells.

The practical application of engineered nanomaterials or nanoparticles like polyamidoamine (PAMAM) dendrimers has been promoted in medical devices or industrial uses. The safety of PAMAM dendrimers needs to be assessed when used as a drug carrier to treat brain disease. However, the effects of PAMAM on the human nervous system remain unknown. In this study, human neural progenitor cells cultured as a 3D neurosphere model were used to study the effects of PAMAM dendrimers on the nervous system. Neurospheres were exposed to different G4-PAMAM dendrimers for 72 h at concentrations of 0.3, 1, 3, and 10 µg/ml. The biodistribution was investigated using fluorescence-labeled PAMAM dendrimers, and gene expression was evaluated using microarray analysis followed by pathway and network analysis. Results showed that PAMAM dendrimer nanoparticles can penetrate into neurospheres via superficial cells on them. PAMAM-NH2 but not PAMAM-SC can inhibit neurosphere growth. A reduced number of MAP2-positive cells in flare regions were inhibited after 10 days of differentiation, indicating an inhibitory effect of PAMAM-NH2 on cell proliferation and neuronal migration. A microarray assay showed 32 dendrimer toxicity-related genes, with network analysis showing 3 independent networks of the selected gene targets. Inducible immediate early gene early growth response gene 1 (Egr1), insulin-like growth factor-binding protein 3 (IGFBP3), tissue factor pathway inhibitor (TFPI2), and adrenomedullin (ADM) were the key genes in each network, and the expression of these genes was significantly down regulated. These findings suggest that exposure of neurospheres to PAMAM-NH2 dendrimers affects cell proliferation and migration through pathways regulated by Egr1, IGFBP3, TFPI2, and ADM.

Effects of Chronic Low-Dose Radiation on Human Neural Progenitor Cells.

The effects of chronic low-dose radiation on human health have not been well established. Recent studies have revealed that neural progenitor cells are present not only in the fetal brain but also in the adult brain. Since immature cells are generally more radiosensitive, here we investigated the effects of chronic low-dose radiation on cultured human neural progenitor cells (hNPCs) derived from embryonic stem cells. Radiation at low doses of 31, 124 and 496 mGy per 72 h was administered to hNPCs. The effects were estimated by gene expression profiling with microarray analysis as well as morphological analysis. Gene expression was dose-dependently changed by radiation. By thirty-one mGy of radiation, inflammatory pathways involving interferon signaling and cell junctions were altered. DNA repair and cell adhesion molecules were affected by 124 mGy of radiation while DNA synthesis, apoptosis, metabolism, and neural differentiation were all affected by 496 mGy of radiation. These in vitro results suggest that 496 mGy radiation affects the development of neuronal progenitor cells while altered gene expression was observed at a radiation dose lower than 100 mGy. This study would contribute to the elucidation of the clinical and subclinical phenotypes of impaired neuronal development induced by chronic low-dose radiation.

Peroxisome proliferator-activated receptor α mediates di-(2-ethylhexyl) phthalate transgenerational repression of ovarian Esr1 expression in female mice.

Di-(2-ethylhexyl)-phthalate (DEHP) is a phthalate ester that binds peroxisome proliferator-activated receptor α (PPARα) to induce proliferation of peroxisomes and regulate the expression of specific target genes. The question of whether the effect of DEHP on female reproductive processes is mediated via PPARα-dependent signaling is controversial. In this study, we investigated the effect of exposure to DEHP on ovarian expression of estrogen receptor α (Esr1) and aromatase (Cyp19a1) in three generations of Sv/129 wild-type (WT, +/+) and PPARα (-/-) knockout mice. Compared with untreated controls, ovarian expression of Esr1 decreased in response to DEHP treatment in the F0 (0.56-fold, P=0.19), F1 (0.45-fold, P=0.023), and F2 (0.35-fold, P=0.014) generations of WT mice, but not PPARα-null mice. Our data indicate that transgenerational repression by DEHP of ovarian Esr1 gene expression is mediated by PPARα-dependent pathways. Further studies are required to elucidate the mechanisms underlying crosstalk between PPARα and Esr1 signaling in reproductive processes.