A Human Stem Cell-Derived Assay for Detection of Toxicants that Promote Obesity

摘要 肥胖是一个重大的全球健康问题。根据《美国医学杂志》最近的一篇报道 协会非西班牙裔黑人经年龄调整后的肥胖率最高(49.5%) 墨西哥裔美国人(40.4%)，所有西班牙裔(39.1%)和非西班牙裔白人(34.3%)。然而，遗传学 无法解释近代史上肥胖症的流行比例。环境毒物影响 干细胞分化的表观遗传学程序。成脂作用是分化程度最高的一种。 对成脂分化的分析和遗传程序进行了很好的描述。许多肥胖症患者 已被证明在表观遗传学上调节脂肪生成。我们建议开发一种简单易用的低温保存 用于销售和经销的致癌试剂盒。该试剂板将作为关键试剂在 用于识别和测量人类生物反应的高通量预测模型的开发 致肥胖的毒物。试剂板将预先种植脂肪生成反应荧光素酶报告- 转导的前脂肪细胞，冷冻保存，运往客户，复温，然后用于直接，快速， 对潜在致肥者的可重复性和敏感度测试。这项技术将有助于确定化学品的优先顺序 更广泛的毒理学评估，支持更多体内生物反应的预测模型，以及 允许评估前脂肪细胞(来源于人类可诱导的多能干细胞，来自患有 不同的遗传或疾病背景)。本阶段SBIR I可行性研究的拟议工作如下 在两个具体目标中执行。在第一个目标中，前脂肪细胞将稳定地转导成脂肪- 由萤火虫荧光素酶检测和Renilla荧光素酶组成的响应性双荧光素酶报告系统 积极的记者。这些克隆将在使用REPLICATE序列的标准成脂检测中受到挑战 两种已确定的致肥性物质的稀释和组合。在Aim 2优化实验中， 96孔法对转导和对照贴壁前脂肪细胞的冷冻保存和解冻 将进行车牌表演。评估方法将包括1)细胞存活和增殖，2)基线 成脂分化；3)对肥胖原的反应(S)。升级到第二阶段的可行性将是 证明&gt；70%的活性，细胞存活并保持对组织培养底物的附着，以及 96孔板解冻后功能性成脂反应报告系统。在第二阶段，我们将评估 利用我们最初的转导克隆和额外的前脂肪细胞克隆的广泛的潜在致肥者 来自不同疾病和遗传背景的个体。我们还将评估其他多井板块 用于更高吞吐量的应用程序的格式，并在必要时进一步修改脂肪生成检测 系统，以提高肥胖发生检测的灵敏度。

ABSTRACT Obesity is a major global health issue. According to a recent report in the Journal of the American Medical Association non-Hispanic blacks have the highest age-adjusted rates of obesity (49.5%) compared with Mexican Americans (40.4%), all Hispanics (39.1%) and non-Hispanic whites (34.3%). However, genetics cannot account for the epidemic proportions of obesity in recent history. Environmental toxicants impact epigenetic programs of stem cell differentiation. Adipogenesis is among the best developed of differentiation assays and the genetic program of adipogenic differentiation is well characterized. Many obesogens have been shown to epigenetically regulate adipogenesis. We propose to develop an easy-to-use cryopreserved obesogenesis assay plate for sale and distribution. This assay plate will serve as a key reagent in the development of high-throughput predictive models for identifying and measuring biological response in humans to 'obesogen' toxicants. The assay plate will be pre-seeded with adipogenesis-responsive luciferase reporter- transduced preadipocytes, cryopreserved, shipped to customers, rewarmed and then used for direct, rapid, reproducible and sensitive testing of potential obesogens. This technology will help to prioritize chemicals for more extensive toxicological evaluation, support more predictive models of in vivo biological response, and permit assessment of preadipocytes (derived from human inducible pluripotent stem cells from individuals with different genetic or disease backgrounds). The proposed work in this Phase SBIR I feasibility study will be performed in 2 specific aims. In the first aim preadipocytes will be stably transduced with an adipogenesis- responsive dual-luciferase reporter system consisting of the 'Firefly' luciferase test and 'Renilla' luciferase positive reporters. These clones will be challenged in a standard adipogenic assay using replicate serial dilutions and combinations of two established obesogens. In aim 2 optimization experiments for the cryopreservation, storage and thawing of transduced and control adherent preadipocytes on 96 well assay plates will be performed. The assessment methods will include 1) cell viability and proliferation, 2) base line adipogenic differentiation, and 3) response to obesogen(s). Feasibility for progression to Phase II will be demonstration of >70% viability, cell survival with retention of attachment to the tissue culture substrate, and functional adipogenesis-responsive reporter system post-thaw in 96 well plates. In Phase II we will assess a broad range of potential obesogens employing our initial transduced clones and additional preadipocyte clones from individuals with different diseases and genetic backgrounds. We will also assess other multi-well plate formats for even higher throughput applications and, if necessary, further modify adipogenesis detection systems to increase the obesogenesis assay sensitivity.