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

 描述(申请人提供)：人脑发育非常容易受到化学物质的影响，因为未成熟的细胞正在积极地分化成更复杂的细胞类型(如神经元、星形胶质细胞、少突胶质细胞)，以形成神经回路。这种脆弱性的一个主要原因是神经元的分化需要对表观基因组进行关键的时间依赖的改变(即神经表观遗传学)，而环境暴露对活跃分化细胞的表观基因组构成了严重的风险。值得注意的是，表观基因组扰动导致的异常大脑被认为是包括自闭症和精神分裂症在内的几种神经疾病的潜在原因。然而，尽管存在这种风险，我们环境中存在的影响人类神经元分化并通过干扰正在发育的表观基因组而做到这一点的化学物质的特性是滞后的。Nzumbe Inc.正在响应RFA-ES-15-005《用于筛选化学毒物对细胞分化的影响的新方法》，应用程序开发一种筛选方法，以识别干扰神经分化过程的化学物质。我们的重点将放在扰乱表观遗传机制的化学物质上，因为它们有可能稳定和异常地影响大脑发育。我们将使用神经元和神经胶质细胞分化的标记来确定化学暴露的影响。提出了两个具体目标。第一个目标是使用来自人iPS细胞的人神经前体/神经干细胞(HNPC)来建立感兴趣的时间点，因为hNPC分化成细胞亚型，概括了正常的人脑发育。这项工作将在96孔板中进行hNPC的增殖和分化，以促进自动筛选。荧光标记抗体 将允许自动筛选，以区分在每个测试时间点出现的不同细胞亚型及其在细胞总数中的相对比例。中提议的工作 目标2将测试五种已知和怀疑具有表观遗传活性的化学物质对它们扰乱正常分化的能力的影响，如目标1所述。这些化学物质包括两种 分别抑制DNA甲基化和组蛋白脱乙酰化的表观遗传抑制剂5-脱氧氮胞苷和曲古抑素A作为初始对照。接下来我们将测试两个 一种是毒素21清单上的化学物质，丙戊酸和双酚A，它可以扰乱表观基因组和改变大脑发育，以及第三种化学物质甲氧基甲醇，它对神经元发育有明显的影响，是毒素21清单上几种化学物质的代谢物和前体。拟议的工作将确定离散的时间点和细胞特定的标记，这些标记可以 用于筛选暴露于具有表观遗传活性的化学品后神经元分化的异常变化。这项工作将导致一种定义明确的分析，可以在第二阶段资助的项目中商业化。

 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.