Regulation of human cortical interneuron development by the chromatin remodeling protein CHD2.

Mutations in the chromodomain helicase DNA binding protein 2 (CHD2) gene are associated with neurodevelopmental disorders. However, mechanisms by which CHD2 regulates human brain development remain largely uncharacterized. Here, we used a human embryonic stem cell model of cortical interneuron (hcIN) development to elucidate its roles in this process. We identified genome-wide CHD2 binding profiles during hcIN differentiation, defining direct CHD2 targets related to neurogenesis in hcIN progenitors and to neuronal function in hcINs. CHD2 bound sites were frequently coenriched with histone H3 lysine 27 acetylation (H3K27ac) and associated with high gene expression, indicating roles for CHD2 in promoting gene expression during hcIN development. Binding sites for different classes of transcription factors were enriched at CHD2 bound regions during differentiation, suggesting transcription factors that may cooperatively regulate stage-specific gene expression with CHD2. We also demonstrated that CHD2 haploinsufficiency altered CHD2 and H3K27ac coenrichment on chromatin and expression of associated genes, decreasing acetylation and expression of cell cycle genes while increasing acetylation and expression of neuronal genes, to cause precocious differentiation. Together, these data describe CHD2 direct targets and mechanisms by which CHD2 prevents precocious hcIN differentiation, which are likely to be disrupted by pathogenic CHD2 mutation to cause neurodevelopmental disorders.

Differences in the Early Development of Human and Mouse Embryonic Stem Cells.

We performed a systematic analysis of gene expression features in early (10-21 days) development of human vs mouse embryonic cells (hESCs vs mESCs). Many development features were found to be conserved, and a majority of differentially regulated genes have similar expression change in both organisms. The similarity is especially evident, when gene expression profiles are clustered together and properties of clustered groups of genes are compared. First 10 days of mESC development match the features of hESC development within 21 days, in accordance with the differences in population doubling time in human and mouse ESCs. At the same time, several important differences are seen. There is a clear difference in initial expression change of transcription factors and stimulus responsive genes, which may be caused by the difference in experimental procedures. However, we also found that some biological processes develop differently; this can clearly be shown, for example, for neuron and sensory organ development. Some groups of genes show peaks of the expression levels during the development and these peaks cannot be claimed to happen at the same time points in the two organisms, as well as for the same groups of (orthologous) genes. We also detected a larger number of upregulated genes during development of mESCs as compared to hESCs. The differences were quantified by comparing promoters of related genes. Most of gene groups behave similarly and have similar transcription factor (TF) binding sites on their promoters. A few groups of genes have similar promoters, but are expressed differently in two species. Interestingly, there are groups of genes expressed similarly, although they have different promoters, which can be shown by comparing their TF binding sites. Namely, a large group of similarly expressed cell cycle-related genes is found to have discrepant TF binding properties in mouse vs human.

Neuronal developmental gene and miRNA signatures induced by histone deacetylase inhibitors in human embryonic stem cells.

Human embryonic stem cells (hESCs) may be applied to develop human-relevant sensitive in vitro test systems for monitoring developmental toxicants. The aim of this study was to identify potential developmental toxicity mechanisms of the histone deacetylase inhibitors (HDAC) valproic acid (VPA), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) relevant to the in vivo condition using a hESC model in combination with specific differentiation protocols and genome-wide gene expression and microRNA profiling. Analysis of the gene expression data showed that VPA repressed neural tube and dorsal forebrain (OTX2, ISL1, EMX2 and SOX10)-related transcripts. In addition, VPA upregulates axonogenesis and ventral forebrain-associated genes, such as SLIT1, SEMA3A, DLX2/4 and GAD2. HDACi-induced expression of miR-378 and knockdown of miR-378 increases the expression of OTX2 and EMX2, which supports our hypothesis that HDACi targets forebrain markers through miR-378. In conclusion, multilineage differentiation in vitro test system is very sensitive for monitoring molecular activities relevant to in vivo neuronal developmental toxicity. Moreover, miR-378 seems to repress the expression of the OTX2 and EMX2 and therefore could be a regulator of the development of neural tube and dorsal forebrain neurons.

Fam40b is required for lineage commitment of murine embryonic stem cells.

FAM40B (STRIP2) is a member of the striatin-interacting phosphatase and kinase (STRIPAK) complex that is involved in the regulation of various processes such as cell proliferation and differentiation. Its role for differentiation processes in embryonic stem cells (ESCs) is till now completely unknown. Short hairpin RNA (shRNA)-mediated silencing of Fam40b expression in ESCs and differentiating embryoid bodies (EBs) led to perturbed differentiation to embryonic germ layers and their derivatives including a complete abrogation of cardiomyogenesis. Pluripotency factors such as Nanog, Oct4 and Sox2 as well as epigenetic factors such as histone acetyltransferase type B (HAT1) and DNA (cytosine-5)-methyltransferase 3-ß (Dnmt3b) were highly upregulated in Fam40b knockdown EBs as compared with control and scrambled EBs. To examine the relevance of Fam40b for development in vivo, Fam40b was knocked down in developing zebrafish. Morpholino-mediated knockdown of Fam40b led to severe abnormalities of the cardiovascular system, including an impaired expression of ventricular myosin heavy chain (vmhc) and of cardiac myosin light chain 2 (cmlc2) in the heart. We identified the gene product of Fam40b in ESCs as a perinuclear and nucleolar protein with a molecular weight of 96 kDa. We conclude that the expression of Fam40b is essential for the lineage commitment of murine embryonic stem cells (mESCs) into differentiated somatic cells via mechanisms involving pluripotency and epigenetic networks.

A tanscriptomics study to elucidate the toxicological mechanism of methylmercury chloride in a human stem cell based in vitro test.

Traditional approaches in evaluating the hazard of drug candidates on the developing offspring are often time-consuming and cost-intensive. Moreover, variations in the toxicological response of different animal species to the tested substance cause severe problems when extrapolating safety dosages for humans. Therefore, more predictive and relevant toxicological systems based on human cell models are required. In the presented study the environmental toxicant methylmercury chloride (MeHgCl), known to cause structural developmental abnormalities in the brain, was used as reference compound to develop a concept contributing to a mechanistic understanding of the toxicity of an investigated substance. Despite the fact, that there are significant data available from animal studies and from poisonings in Japan and Iraq, uncertainties on the mechanism of MeHgCl during human development are still remaining and qualify the substance for further analysis. Transcriptomics analysis in combination with a human cell based in vitro model has been used in order to elucidate the toxicity of MeHgCl at molecular level. Differentiating neural precursor cells that have been exposed continuously to non- and low-cytotoxic concentrations of MeHgCl were investigated. Quantitative change in the mRNA expression profiles of selected genes demonstrated the sensitivity of the cell model and its qualification for a transcriptomics study screening changes in the expression profile of the complete human genome of MeHgCl-treated human neural cells. Potential biomarkers were identified and these candidate marker genes as well as their involvement in a possible toxic mechanism of MeHgCl during the human neurulation process are hereby introduced. The study confirmed the hypothesis that a cellular model based on a human stem cell line can be applied for elucidating unknown mode of actions of developmental toxicants.

Characterisation of a neural teratogenicity assay based on human ESCs differentiation following exposure to valproic acid.

The development of in vitro testing strategies for chemical and drug screening is a priority need in order to protect human health, to increase safety, to reduce the number of animals required for conventional testing methods and finally to meet the deadlines of current legislations. The aim of this work was to design an alternative testing method based on human embryonic stem cells for the detection of prenatal neural toxicity. For this purpose we have created a model based on the generation of neural rosettes, reproducing in vitro the gastrulation events recapitulating the formation of the neural tube in vivo. To validate the model we have exposed this complex cell system to increasing concentrations of valproic acid, a known teratogenic agent, to analyse the morphological and molecular changes induced by the toxicant. Specific assays were applied to discriminate between cytotoxicity and specific neural toxicity. Transcriptomic analysis was performed with a microarray Affimetrix platform and validated by quantitative real time RT-PCR for the expression of genes involved in early neural development, neural tube formation and neural cells migration, key biological processes in which the effect of valproic acid is most relevant. The results demonstrated that neural rosette cells respond to valproic acid exposure with molecular and morphological changes similar to those observed in vivo, indicating that this method represents a promising alternative test for the detection of human prenatal neural toxicity.

Cytosine arabinoside induces ectoderm and inhibits mesoderm expression in human embryonic stem cells during multilineage differentiation.

BACKGROUND AND PURPOSE: Teratogenic substances induce adverse effects during the development of the embryo. Multilineage differentiation of human embryonic stem cells (hESCs) mimics the development of the embryo in vitro. Here, we propose a transcriptomic approach in hESCs for monitoring specific toxic effects of compounds as an alternative to traditional time-consuming and cost-intensive in vivo tests requiring large numbers of animals. This study was undertaken to explore the adverse effects of cytosine arabinoside (Ara-C) on randomly differentiated hESCs. EXPERIMENTAL APPROACH: Human embryonic stem cells were used to investigate the effects of a developmental toxicant Ara-C. Sublethal concentrations of Ara-C were given for two time points, day 7 and day 14 during the differentiation. Gene expression was assessed with microarrays to determine the dysregulated transcripts in presence of Ara-C. KEY RESULTS: Randomly differentiated hESCs were able to generate the multilineage markers. The low concentration of Ara-C (1 nM) induced the ectoderm and inhibited the mesoderm at day 14. The induction of ectodermal markers such as MAP2, TUBB III, PAX6, TH and NESTIN was observed with an inhibition of mesodermal markers such as HAND2, PITX2, GATA5, MYL4, TNNT2, COL1A1 and COL1A2. In addition, no induction of apoptosis was observed. Gene ontology revealed unique dysregulated biological process related to neuronal differentiation and mesoderm development. Pathway analysis showed the axon guidance pathway to be dysregulated. CONCLUSIONS AND IMPLICATIONS: Our results suggest that hESCs in combination with toxicogenomics offer a sensitive in vitro developmental toxicity model as an alternative to traditional animal experiments.