Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the ‘human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)’ European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (<20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large ‘common response’ to VPA and MeHg could be distinguished from ‘compound-specific’ responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-012-0967-3) contains supplementary material, which is available to authorized users.

Assessment of the network of toxicity pathways by Omics technologies and bioinformatic data processing paves the road toward a new toxicology for the twenty-first century. Especially, the upstream network of responses, taking place in toxicant-treated cells before a point of no return is reached, is still little explored. We studied the effects of the model neurotoxicant 1-methyl-4-phenylpyridinium (MPP+) by a combined metabolomics (mass spectrometry) and transcriptomics (microarrays and deep sequencing) approach to provide unbiased data on earliest cellular adaptations to stress. Neural precursor cells (LUHMES) were differentiated to homogeneous cultures of fully postmitotic human dopaminergic neurons, and then exposed to the mitochondrial respiratory chain inhibitor MPP+ (5 μM). At 18–24 h after treatment, intracellular ATP and mitochondrial integrity were still close to control levels, but pronounced transcriptome and metabolome changes were seen. Data on altered glucose flux, depletion of phosphocreatine and oxidative stress (e.g., methionine sulfoxide formation) confirmed the validity of the approach. New findings were related to nuclear paraspeckle depletion, as well as an early activation of branches of the transsulfuration pathway to increase glutathione. Bioinformatic analysis of our data identified the transcription factor ATF-4 as an upstream regulator of early responses. Findings on this signaling pathway and on adaptive increases of glutathione production were confirmed biochemically. Metabolic and transcriptional profiling contributed complementary information on multiple primary and secondary changes that contribute to the cellular response to MPP+. Thus, combined ‘Omics' analysis is a new unbiased approach to unravel earliest metabolic changes, whose balance decides on the final cell fate.

Exposure to the antiepileptic drug valproic acid (VPA) during gestation causes neurofunctional and anatomic deficits in later life. At present, there are little human data on how early neural development is affected by chemicals. We used human embryonic stem cells, differentiating to neuroectodermal precursors, as a model to investigate the modes of action of VPA. Microarray expression profiling, qPCR of specific marker genes, immunostaining and the expression of green fluorescent protein under the control of the promoter of the canonical neural precursor cell marker HES5 were used as readouts. Exposure to VPA resulted in distorted marker gene expression, characterized by a relative increase in NANOG and OCT4 and a reduction in PAX6. A similar response pattern was observed with trichostatin A, a potent and specific histone deacetylase inhibitor (HDACi), but not with several other toxicants. Differentiation markers were disturbed by prolonged, but not by acute treatment with HDACi, and the strongest disturbance of differentiation was observed by toxicant exposure during early neural fate decision. The increased acetylation of histones observed in the presence of HDACi may explain the up-regulation of some genes. However, to understand the down-regulation of PAX6 and the overall complex transcript changes, we examined further epigenetic markers. Alterations in the methylation of lysines 4 and 27 of histone H3 were detected in the promoter region of PAX6 and OCT4. The changes in these activating and silencing histone marks provide a more general mechanistic rational for the regulation of developmentally important genes at non-cytotoxic drug concentrations.

IMPORTANCE Three-dimensional cerebral organoids generated from patient-derived induced pluripotent stem cells (iPSCs) may be used to interrogate cellular-molecular underpinnings of schizophrenia.OBJECTIVE To determine transcriptomic profiles and functional characteristics of cerebral organoids from patients with schizophrenia using gene expression studies, complemented with investigations of mitochondrial function through measurement of real-time oxygen consumption rate, and functional studies of neuronal firing with microelectrode arrays. DESIGN, SETTING, AND PARTICIPANTSThis case-control study was conducted at Massachusetts General Hospital between 2017 and 2019. Transcriptomic profiling of iPSC-derived cerebral organoids from 8 patients with schizophrenia and 8 healthy control individuals was undertaken to identify cellular pathways that are aberrant in schizophrenia. Induced pluripotent stem cells and cerebral organoids were generated from patients who had been diagnosed as having schizophrenia and from heathy control individuals.MAIN OUTCOMES AND MEASURES Transcriptomic analysis of iPSC-derived cerebral organoids from patients with schizophrenia show differences in expression of genes involved in synaptic biology and neurodevelopment and are enriched for genes implicated in schizophrenia genome-wide association studies (GWAS). RESULTSThe study included iPSC lines generated from 11 male and 5 female white participants, with a mean age of 38.8 years. RNA sequencing data from iPSC-derived cerebral organoids in schizophrenia showed differential expression of genes involved in synapses, in nervous system development, and in antigen processing. The differentially expressed genes were enriched for genes implicated in schizophrenia, with 23% of GWAS genes showing differential expression in schizophrenia and control organoids: 10 GWAS genes were upregulated in schizophrenia organoids while 15 GWAS genes were downregulated. Analysis of the gene expression profiles suggested dysregulation of genes involved in mitochondrial function and those involved in modulation of excitatory and inhibitory pathways. Studies of mitochondrial respiration showed lower basal consumption rate, adenosine triphosphate production, proton leak, and nonmitochondrial oxygen consumption in schizophrenia cerebral organoids, without any differences in the extracellular acidification rate. Microelectrode array studies of cerebral organoids showed no differences in baseline electrical activity in schizophrenia but revealed a diminished response to stimulation and depolarization.CONCLUSIONS AND RELEVANCE Investigations of patient-derived cerebral organoids in schizophrenia revealed gene expression patterns suggesting dysregulation of a number of pathways in schizophrenia, delineated differences in mitochondrial function, and showed deficits in response to stimulation and depolarization in schizophrenia.

Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2), that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide.