A screening assay for chemicals that affect the differentiation of human neural cells.

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 DESCRIPTION (provided by applicant): Human brain development represents a highly vulnerable time to chemical exposures because immature cells are actively undergoing differentiation into more complex cell types (e.g., neurons, astrocytes, oligodendroglia) to form neural circuits. A major reason for this vulnerability is that neuronal differentiation requires critical time-dependent changes to the epigenome (i.e., neuroepigenetics), and environmental exposures pose acute risks to the epigenomes of actively differentiating cells. Significantly, abnormal brain develop from epigenomic perturbation is believed to be an underlying cause of several neurological disorders including autism and schizophrenia. Despite this risk, however, the identity of chemicals present in our environment that affect human neuronal differentiation and do so by perturbing developing epigenomes is lagging. Nzumbe Inc. is responding to RFA-ES-15-005 entitled "Novel Assays for Screening the Effects of Chemical Toxicants on Cell Differentiation" with an application to develop a screening assay to identify chemicals that interfere with the process of neural differentiation. Our focus will be on chemicals that perturb epigenetic mechanisms because of their potential to stably and aberrantly affect brain development. We will use markers of neuronal and glial cell differentiation to identify effects from chemical exposures. Two specific aims are proposed. The first aim will use human neuroprogenitor/neural stem cells (hNPCs) derived from human iPS cells to establish time points of interest as the hNPCs differentiate into cell subtypes recapitulating normal human brain development. This work will be performed with hNPCs undergoing proliferation and differentiation in 96-well plates to facilitate automated screening. Fluorescent-labeled antibodies will allow for automated screening to distinguish the different cell subtypes that arise at each time point tested and their relative proportions in the total cell population. The work proposed in aim 2 will test the effects of five chemicals with known and suspected epigenetic activities for their ability to perturb normal differentiation, as described in aim 1. These chemicals include two well- characterized epigenetic inhibitors, 5-deoxyazacytdine and trichostatin A, which inhibit DNA methylation and histone deacetylation, respectively, as initial controls. Next we will test two chemicals on the tox-21 list, valproic acid and Bisphenol A, which can perturb epigenomes and alter brain development, and a third chemical, methylazoxymethanol, which has clear effects on neuronal development and is both a metabolite and precursor of several chemicals on the tox-21 list. The proposed work will identify discrete time points and cell-specific markers that can be used to screen for aberrant changes in neuronal differentiation following exposures to chemicals with epigenetic activities. This work will lead to a well-defined assay that can be commercialized in a phase II funded program.