RAPID NEURAL DIFFERENTIATION OF HUMAN STEM CELLS: A NOVEL DRUG DISCOVERY PLATFORM

描述（由申请人提供）：制药行业目前面临的主要挑战是开发脑部疾病和精神障碍的治疗方法，如抑郁症、焦虑症、精神分裂症、癫痫、成瘾等。在过去的十年中，随着技术的不断进步，药物发现过程发生了巨大的变化。除了通过分析小型化和多路复用来提高高通量筛选（HTS）的效率外，还采取了各种举措来改进预测毒性的科学和改进对人类的推断。随着基因组学和蛋白质组学的最新进展，许多新技术使得利用具有过表达药物靶点的活细胞进行检测成为药物发现过程中的一个重要方面。然而，一个合适的细胞平台的HTS神经目标是不可用的。这在一定程度上可归因于在培养中转染和维持神经元的困难。最近，人类胚胎干细胞研究取得了突破，可以从可再生的祖细胞来源中体外分化神经元。然而，目前分化方案的局限性和较差的神经祖细胞转染技术限制了这些细胞在试验中的使用。Originus公司可以通过使用专有的固相细胞转染技术（称为表面转染和表达协议（STEP））来解决这些问题，从而有助于开发复杂的基于神经元细胞的检测平台。我们已经成功地开发了多个G蛋白偶联受体（GPCR）的测定在神经和非神经细胞系使用STEP。提出的新的STEP转染和分化平台将大大加速人类干细胞神经分化过程，同时引入感兴趣的目标和必要的成分，用于基于细胞的检测。我们的目标不仅是开发更可靠和强大的分析方法，以提高早期临床前阶段的筛查效率，而且还开发更多与生理相关的分析方法，以提高临床成功率。在这个应用程序的第一阶段，我们将在实验中初步检查神经祖细胞的生物学反应，并彻底表征由选定的基本螺旋-环-螺旋（bHLH）转录因子的短暂过表达诱导的祖细胞神经分化。此外，这种基于细胞的检测平台的潜在意义将通过检查分化的人类神经元中代谢性谷氨酸受体的反应来证明。在二期研究中，我们计划将这项技术应用于其他人类多能细胞系，扩大bHLH因子的测试范围，以微调分化过程，并进一步开发针对不同药物靶点的检测方法。该平台将使药物靶点在人类神经元中表达的HTS成为可能。此外，它还可用于毒理学、神经分化和变性研究。公共卫生相关性：拟议的研究旨在通过利用人类干细胞和神经元发育遗传控制的研究进展，大大提高脑疾病和精神障碍药物发现过程的有效性。具体来说，本应用程序的目标是为胚胎干细胞衍生的人类神经元开发生理学相关的药物筛选平台。

DESCRIPTION (provided by applicant): A current major challenge in pharmaceutical industry is to develop treatments for brain diseases and mental disorders such as depression, anxiety, schizophrenia, epilepsy, addiction and many more. Over the last decade, the drug discovery process has evolved dramatically following the continued advancement of technologies. In addition to increasing the efficiency of high-throughput screening (HTS) by assay miniaturization and multiplexing, various initiatives have been taken to improve the science of predicting toxicity and improving extrapolation to humans. With recent advances in genomics and proteomics, a number of novel technologies have made the utilization of live cells with overexpressed drug targets in assays an important aspect in the drug discovery process. Nevertheless, an appropriate cellular platform for HTS of neural targets has not been available. This in part can be attributed to difficulties in transfecting and maintaining neurons in culture. Recently, a breakthrough was made in human embryonic stem cell research that allows in vitro differentiation of neurons from a renewable source of progenitor cells. However, limitations of the current differentiation protocol and poor neuroprogenitor cell transfection techniques have restricted the use of these cells in assays. Originus, Inc. can contribute to the development of sophisticated neuronal cell-based assay platforms that address these issues by using a proprietary solid phase cell transfection technology termed Surface Transfection and Expression Protocol (STEP) exclusively licensed from the University of Michigan. We have successfully developed multiple G- protein-coupled receptor (GPCR) assays in neuronal and non-neuronal cell lines using STEP. The proposed new STEP transfection and differentiation platform will drastically accelerate human stem cell neural differentiation process while simultaneously introducing the target of interest and necessary components for a cell-based assay. Our goal is to develop not only more reliable and robust assays that increase the screening efficiency in early preclinical phase, but also more physiologically relevant assays that may boost clinical success rates. During Phase I of this application we will initially examine the biological responses of the neural progenitor cells in assays, and thoroughly characterize the progenitor cell neuronal differentiation induced by transient over-expression of selected basic helix-loop-helix (bHLH) transcription factors. Further, the potential implication of such a platform for cell-based assays will be demonstrated by examining the response of metabotropic glutamate receptors in differentiated human neurons. During the Phase II, we plan to apply this technology to other human pluripotent cell lines, expand the spectrum of bHLH factors tested to fine tune the differentiation process, and to further develop assays for different drug targets. This novel platform will make feasible HTS of drug targets expressed in human neurons. In addition, it may also be used for toxicology, neural differentiation and degeneration studies. PUBLIC HEALTH RELEVANCE: The proposed studies aim to greatly improve the effectiveness of the drug discovery process for brain diseases and mental disorders by taking advantage of research advances in human stem cells and genetic control of neuronal development. Specifically, the goal of this application is to develop physiologically relevant drug screening platforms for human neurons derived from embryonic stem cells.