Basement membrane (BM) is a compact and continuous sheet of 50–350 nm thickness and located underneath epithelium, endothelium, etc. It helps tissue repair and morphogenesis, and stem cells' differentiation to mature phenotypes. We have devised several culture models for cellular assembly of BM in vitro and developed a thorough removal of the covering BM-forming cells in order to utilize the bared BM as a culture substratum. Thus de novo synthesized BM (sBM) substrata of LN-511 isoform normally guided tissue progenitors or embryonic stem cells to terminal differentiation and morphologically and functionally mature phenotypes, for example, airway ciliated cells, hepatocytes and pancreatic β cells. Therefore sBM has been proven to be a matrix, that is, solid-phase cradle. BM formation, which we introduce in this chapter, has the advantages of it not being necessary to prepare each BM component, but also being free from clearing irregular aggregates that come from malassembly among them. We simply culture epithelial and endothelial cells etc. on the fibrillar collagen substratum that has been coated beforehand with N-acetylglucosamine ligands-branching or -sprouting hydrophobic polymer, and wait for automatic assembly of BM structure by these cells. rLN-10 cells, genetically modified cells, are advantageous for forming an optimized sBM.

BACKGROUND:The neurological effects of short-term dioxin exposure during the fetal period is an important health risk in humans. Here, we investigated the effects of dioxin on neural differentiation using human embryonic stem cells (hESCs) to evaluate human susceptibility to dioxin.METHODS:Using an enzymatic bulk passage, neural differentiation from human ESCs was carried out. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was added to various stages of culture. The expression levels of the neuronal markers microtubule-associated protein 2 (MAP2) and thyroxine hydroxylase (TH) were measured by RT-qPCR and image analysis of immunostaining.RESULTS:Although early-stage neuronal cells are quite resistant to TCDD, the numbers of neural rosettes and increases in mRNA expression levels and the number of cells positive for MAP2 and TH were significant by temporal exposure at embryoid body stage (Day9-exposure group). In contrast, the TCDD exposures against ESCs (Day0-exposure group) and differentiated neural cells (Day35-exposure group) were not affected at all. The increment was similarly observed by continuous exposure of TCDD from Day9 through Day60.CONCLUSIONS:These results indicated that dioxin exposure during the early stage of differentiation from hESCs increases the contents of neuronal cells, especially TH-positive neuronal cells. Regulations of aryl hydrocarbon receptor (AHR) signaling in an early stage of embryogenesis should be investigated extensively to understand the mechanism underlying the increase in neuronal cell populations and to apply the knowledge to regenerative medicine.

Toxicity prediction based on stem cells and tissue derived from stem cells plays a very important role in the fields of biomedicine and pharmacology. Here we report on qRT-PCR data obtained by exposing 20 compounds to human embryonic stem (ES) cells. The data are intended to improve toxicity prediction, per category, of various compounds through the use of support vector machines, and by applying gene networks. The accuracy of our system was 97.5-100% in three toxicity categories: neurotoxins (NTs), genotoxic carcinogens (GCs), and non-genotoxic carcinogens (NGCs). We predicted that two uncategorized compounds (bisphenol-A and permethrin) should be classified as follows: bisphenol-A as a non-genotoxic carcinogen, and permethrin as a neurotoxin. These predictions are supported by recent reports, and as such constitute a good outcome. Our results include two important features: 1) The accuracy of prediction was higher when machine learning was carried out using gene networks and activity, rather than the normal quantitative structure-activity relationship (QSAR); and 2) By using undifferentiated ES cells, the late effect of chemical substances was predicted. From these results, we succeeded in constructing a highly effective and highly accurate system to predict the toxicity of compounds using stem cells.

Predictive toxicology using stem cells or their derived tissues has gained increasing importance in biomedical and pharmaceutical research. Here, we show that toxicity category prediction by support vector machines (SVMs), which uses qRT-PCR data from 20 categorized chemicals based on a human embryonic stem cell (hESC) system, is improved by the adoption of gene networks, in which network edge weights are added as feature vectors when noisy qRT-PCR data fail to make accurate predictions. The accuracies of our system were 97.5-100% for three toxicity categories: neurotoxins (NTs), genotoxic carcinogens (GCs) and non-genotoxic carcinogens (NGCs). For two uncategorized chemicals, bisphenol-A and permethrin, our system yielded reasonable results: bisphenol-A was categorized as an NGC, and permethrin was categorized as an NT; both predictions were supported by recently published papers. Our study has two important features: (i) as the first study to employ gene networks without using conventional quantitative structure-activity relationships (QSARs) as input data for SVMs to analyze toxicogenomics data in an hESC validation system, it uses additional information of gene-to-gene interactions to significantly increase prediction accuracies for noisy gene expression data; and (ii) using only undifferentiated hESCs, our study has considerable potential to predict late-onset chemical toxicities, including abnormalities that occur during embryonic development.

The establishment of more efficient in vitro approaches has been widely acknowledged as a critical need for toxicity testing. In this study, we examined the effects of methylmercury (MeHg), which is a well-known developmental neurotoxicant, in two neuronal differentiation systems of mouse and human embryonic stem cells (mESCs and hESCs, respectively). Embryoid bodies were generated from gathering of mESCs and hESCs using a micro-device and seeded onto ornithine-laminin-coated plates to promote proliferation and neuronal differentiation. The cells were exposed to MeHg from the start of neuronal induction until the termination of cultures, and significant reductions of mESCs and hESCs were observed in the cell viability assays at 1,10,100 and 1000nM, respectively. Although the mESC derivatives were more sensitive than the hESC derivatives to MeHg exposure in terms of cell viability, the morphological evaluation demonstrated that the neurite length and branch points of hESC derivatives were more susceptible to a low concentration of MeHg. Then, the mRNA levels of differentiation markers were examined using quantitative RT-PCR analysis and the interactions between MeHg exposure and gene expression levels were visualized using a network model based on a Bayesian algorithm. The Bayesian network analysis showed that a MeHg-node was located on the highest hierarchy in the hESC derivatives, but not in the mESC derivatives, suggesting that MeHg directly affect differentiation marker genes in hESCs. Taken together, effects of MeHg were observed in our neuronal differentiation systems of mESCs and hESCs using a combination of morphological and molecular markers. Our study provided possible, but limited, evidences that human ESC models might be more sensitive in particular endpoints in response to MeHg exposure than that in mouse ESC models. Further investigations that expand on the findings of the present paper may solve problems that occur when the outcomes from laboratory animals are extrapolated for human risk evaluation.

The establishment of more efficient approaches for developmental neurotoxicity testing (DNT) has been an emerging issue for children's environmental health. Here we describe a systematic approach for DNT using the neuronal differentiation of mouse embryonic stem cells (mESCs) as a model of fetal programming. During embryoid body (EB) formation, mESCs were exposed to 12 chemicals for 24 h and then global gene expression profiling was performed using whole genome microarray analysis. Gene expression signatures for seven kinds of gene sets related to neuronal development and neuronal diseases were selected for further analysis. At the later stages of neuronal cell differentiation from EBs, neuronal phenotypic parameters were determined using a high-content image analyzer. Bayesian network analysis was then performed based on global gene expression and neuronal phenotypic data to generate comprehensive networks with a linkage between early events and later effects. Furthermore, the probability distribution values for the strength of the linkage between parameters in each network was calculated and then used in principal component analysis. The characterization of chemicals according to their neurotoxic potential reveals that the multi-parametric analysis based on phenotype and gene expression profiling during neuronal differentiation of mESCs can provide a useful tool to monitor fetal programming and to predict developmentally neurotoxic compounds.

We have previously established a protocol for the neural differentiation of mouse embryonic stem cells (mESCs) as an efficient tool to evaluate the neurodevelopmental toxicity of environmental chemicals. Here, we described a multivariate bioinformatic approach to identify the stage-specific gene sets associated with neural differentiation of mESCs. We exposed mESCs (B6G-2 cells) to 10(-8) or 10(-7) M of retinoic acid (RA) for 4 days during embryoid body formation and then performed morphological analysis on day of differentiation (DoD) 8 and 36, or genomic microarray analysis on DoD 0, 2, 8, and 36. Three gene sets, namely a literature-based gene set (set 1), an analysis-based gene set (set 2) using self-organizing map and principal component analysis, and an enrichment gene set (set 3), were selected by the combined use of knowledge from literatures and gene information selected from the microarray data. A gene network analysis for each gene set was then performed using Bayesian statistics to identify stage-specific gene expression signatures in response to RA during mESC neural differentiation. Our results showed that RA significantly increased the size of neurosphere, neuronal cells, and glial cells on DoD 36. In addition, the gene network analysis showed that glial fibrillary acidic protein, a neural marker, remarkably up-regulates the other genes in gene set 1 and 3, and Gbx2, a neural development marker, significantly up-regulates the other genes in gene set 2 on DoD 36 in the presence of RA. These findings suggest that our protocol for identification of developmental stage-specific gene expression and interaction is a useful method for the screening of environmental chemical toxicity during neurodevelopmental periods.

Thalidomide is increasingly used in anticancer and anti-inflammation therapies. However, it is known for its teratogenicity and ability to induce peripheral neuropathy, although the mechanisms underlying its neurological effect in humans are unclear. In this study, we investigated the effect of thalidomide on the metabolism and neuronal differentiation of human neural progenitor cells. We found that levels of tyrosine, phenylalanine, methionine and glutathione, which are involved in dopamine and methionine metabolism, were decreased following thalidomide treatment. Morphological analysis revealed that treatment with 100 nM thalidomide, which is much lower than clinical doses, significantly decreased the number of dopaminergic (tyrosine hydroxylase-positive) neurons, compared with control cells. Our results suggest that these adverse neurological effects of thalidomide should be taken into consideration prior to its use for the treatment of neurodegenerative and other diseases.

Pyrethroids are one of the most widely used classes of insecticides and show neurotoxic effects that induce oxidative stress in the neonatal rat brain. However, little is still known about effects of prenatal exposure to permethrin on vascular development in fetal brain, central nervous system development, and adult offspring behaviors. In this study, the effects of prenatal exposure to permethrin on the development of cerebral arteries in fetal brains, neurotransmitter in neonatal brains, and locomotor activities in offspring mice were investigated. Permethrin (0, 2, 10, 50, and 75 mg/kg) was orally administered to pregnant females once on gestation day 10.5. The brains of permethrin-treated fetuses showed altered vascular formation involving shortened lengths of vessels, an increased number of small branches, and, in some cases, insufficient fusion of the anterior communicating arteries in the area of circle of Willis. The prenatal exposure to permethrin altered neocortical and hippocampus thickness in the mid brain and significantly increased norepinephrine and dopamine levels at postnatal day 7 mice. For spontaneous behavior, the standing ability test using a viewing jar and open-field tests showed significant decrease of the standing ability and locomotor activity in male mice at 8 or 12 weeks of age, respectively. The results suggest that prenatal exposure to permethrin may affect insufficient development of the brain through alterations of vascular development.

Environmental chemicals with estrogenic activity, known as xenoestrogens, may cause impaired reproductive development and endocrine-related cancers in humans by disrupting endocrine functions. Aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) is believed to play important roles in a variety of physiological processes, including estrogen signaling pathways, that may be involved in the pathogenesis and therapeutic responses of endocrine-related cancers. However, much of the underlying mechanism remains unknown. In this study, we investigated whether ARNT2 expression is regulated by a range of representative xenoestrogens in human cancer cell lines. Bisphenol A (BPA), benzyl butyl phthalate (BBP), and 1,1,1-trichloro-2,2-bis(2-chlorophenyl-4-chlorophenyl)ethane (o,p'-DDT) were found to be estrogenic toward BG1Luc4E2 cells by an E-CALUX bioassay. ARNT2 expression was downregulated by BPA, BBP, and o,p'-DDT in a dose-dependent manner in estrogen receptor 1 (ESR1)-positive MCF-7 and BG1Luc4E2 cells, but not in estrogen receptor-negative LNCaP cells. The reduction in ARNT2 expression in cells treated with the xenoestrogens was fully recovered by the addition of a specific ESR1 antagonist, MPP. In conclusion, we have shown for the first time that ARNT2 expression is modulated by xenoestrogens by an ESR1-dependent mechanism in MCF-7 breast cancer cells.

Profiles of Chemical Effects on Cells (pCEC) is a toxicogenomics database with a system of classifying chemicals that have effects on human health. This database stores and handles gene expression profiling information and categories of toxicity data. Chemicals are classified according to the specific tissues and cells they affect, the gene expression changes they induce, their toxicity and biological functions in this database system. The pCEC system also analyzes relationships between chemicals and the genes they affect in specific tissues and cells. The reason why we developed pCEC is to support decision-making within the context of environmental regulation. Especially, exposure to environmental chemicals during fetal and newborn development may result in a predisposition to various disorders such as cancer, learning disabilities and allergies later in life. The identification and prediction of hazardous chemicals using limited information are important issues in human health risk management. Therefore, various toxicity information including lethal dose 50 (LD50), toxicity pathways and pathological data were loaded into pCEC. pCEC is also a facility for query, analysis and prediction of unknown toxicochemical reaction pathways and biomarkers which are based on toxicoinformatical data mining approaches. This database is available online at http://project.nies.go.jp/eCA/cgi-bin/index.cgi. The current version of the database has information on the hepatotoxicity, reproductive toxicity and embryotoxicity of chemicals.

The sudden infant death syndrome (SIDS) is still the main cause of postneonatal infant death and its cause is still unknown. A chronic hypoxic situation has been shown to exist in the brains of SIDS victims and apoptosis has been demonstrated in hypoxic situations. In this study, the correlation between apoptotic neurons or glias and sleep apnea in SIDS was investigated in the brainstem of SIDS victims. Materials and methods: In a cohort of 27,000 infants studied prospectively to characterize their sleep–wake behavior, 38 infants died under 6 months of age. They included 26 cases of SIDS. The frequency and duration of sleep apnea were analyzed. The brainstem material was collected and terminal-deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method was carried out. The density of TUNEL-positive neurons or glias was measured quantitatively. Correlation analyses were carried out between the apoptosis-associated pathological data and the physiological data of sleep apnea. Results: No significant negative or positive correlation between the density of TUNEL-positive neurons or glias and the characteristics of sleep apnea was observed in SIDS victims. No statistically significant differences associated with apoptotic neurons and glias were observed between SIDS and non-SIDS. Conclusions: The pathological findings of apoptosis were not in agreement with the hypothesis refer to apnea and arousal phenomenon in pathophysiology of SIDS.

TCDD (2,3,7,8-tetrachlorodebenzo-p-dioxin) requires the presence of the aryl hydrocarbon receptor (Ahr) gene for its toxic effects, such as reproductive disorders in male offspring of maternally exposed rats and mice. To study the involvement of the Ahr gene in producing the toxic phenotype with respect to testicular development, we administered a relatively high dose of TCDD to mice with three different maternally derived Ahr genotypic traits, and then compared several Ahr-dependent alterations among male reproductive systems on Postnatal Day 14. Reduction in anogenital distance and expression of prostatic epithelial genes in the urogenital complex (UGC) were detected in Ahr(+/+) and Ahr(+/-) mice exposed to TCDD, whereas no difference was observed in Ahr(-/-) mice. In situ hybridization revealed the absence of probasin mRNA expression in the prostate epithelium, despite the obvious development of prostatic lobes in TCDD-exposed mice. In contrast to obvious prostatic dysfunction and induction of cytochrome P450 (CYP) family genes in the UGC by TCDD, no alterations in testicular functions were observed in germ cell/Sertoli cell/interstitial cell marker gene expression or CYP family induction. No histopathological changes were observed among the three genotypes and between control and TCDD-exposed mice. Therefore, mouse external genitalia and prostatic development are much more sensitive to TCDD treatment than testis. Further, the Ahr gene, analyzed in this study, does not significantly contribute to testicular function during perinatal and immature stages, and the developing mouse testis appears to be quite resistant to TCDD exposure.

To determine a site producing a low molecular weight haemagglutinin isolated from the miniature swine sperm surface, Western blot and immunohistochemistry with boar tissue extracts and paraffin sections were employed. Using anti-13K and anti-16K sera raised against 13K and 16K-proteins purified by electroelution, immunoblotting clearly showed that out of six different tissue extracts and two secretory fluids, only seminal vesicular extract and seminal vesicular fluid were positive to these antibodies. Immunohistochemistry also demonstrated that the cytoplasm of epithelial cells of seminal vesicle and secretory materials seen above the epithelial cells were strongly stained with these antisera. The present data indicated that 13K and 16K-proteins were synthesized in the seminal vesicle and then bound to sperm surface in male reproductive tracts.

To investigate seminal plasma factors which damage the surface of the sperm head during freezing treatment, proteins were recovered from the surface of ejaculated spermatozoa of miniature swine. Semen of miniature swine was washed with phosphate buffered saline and treated with hypertonic saline solutions. The fraction showed significant haemagglutination activity. Proteins in the fraction were separated by SDS-PAGE. Five major proteins were purified by excision from the gels followed by electro-elution, and then antisera against the proteins were produced using mice. Haemagglutination inhibitory assay demonstrated that only anti-13K serum inhibited haemagglutination of the fraction. Immunocytochemistry clearly showed that anti-13K serum reacted to the whole surface of ejaculated sperm. Furthermore the antiserum showed a slightly stronger reaction to the swollen surface of one-hour frozen spermatozoa than untreated spermatozoa. The materials seen in the frozen sperm specimen, considered as aggregates of the released sperm surface complex, also reacted with the anti-13K serum intensively. In addition, the 13K-protein was constantly detected in seminal plasma precipitates during the entire freezing treatment. The present data indicated that one of the sperm surface proteins isolated with a hypertonic saline solution is a haemagglutinin, which might be involved in the aggregation of seminal plasma during freezing.