Revolutionary Technology for Efficient Derivation of Human iPSCs with Messenger R

描述（由申请人提供）：拟议项目的目标是开发一种商业试剂盒，使正在进行项目的药物成瘾研究人员能够使用基于细胞的模型成功和方便地生产人类诱导多能干细胞（iPSCs）。药物成瘾摧毁了药物滥用者及其家庭的生活，造成了巨大的社会问题，并增加了国家医疗保健系统的成本。导致成瘾开始和建立的分子机制在很大程度上是未知的，部分原因是缺乏来自患者的神经细胞类型。使人类多能干细胞成为可能的技术，通过使用患者特异性细胞来源，为直接询问成瘾病理生理学的新领域打开了大门。目前，NIDA正在资助依赖iPS技术的项目：1)表征来自具有基因组畸变的iPSCs的神经元的多巴胺能神经传递功能，例如包含多巴胺转运蛋白（DAT）和囊泡单胺转运蛋白（VMAT2）基因的染色体多态性区域；2)通过iPSCs鉴定与患者细胞源性神经元成瘾功能相关的遗传变异；3)从患者iPSCs中提取多巴胺神经元，并将其暴露于甲基苯丙胺和戒断中，以分析患者特异性神经元与对照组的电生理特性等。尽管它被认为是一项在成瘾或其他疾病研究中具有巨大潜力的技术，但对大多数研究人员来说，将成人细胞重新编程为多能干细胞仍然是一个低效、繁琐和昂贵的过程。目前流行的重编程方法还有其他问题，如基因组的改变或病毒的引入，这些问题可能使基于细胞的分析结果的解释复杂化。该项目旨在开发一种交钥匙工具包，使许多生物医学研究人员能够在不到两周的时间内以比其他方法高10到100倍的效率生成iPSCs，而且通过无饲料、无异种和无足迹的过程。

DESCRIPTION (provided by applicant): The goal of the proposed project is to develop a commercial kit that will allow drug addiction researchers with ongoing projects using cell-based models to be able to successfully and conveniently produce human induced pluripotent stem cells (iPSCs). Addiction to drugs destroys lives of substance abusers and their families, creating enormous social problems and exerting cost on the nation's health care system. Molecular mechanisms that contribute to the initiation and establishment of addiction are largely unknown, partially due to the lack of neural cell types from the patients. The technologies that made possible of human iPSCs opened the doors to new areas of direct interrogation of the pathophysiology of addiction by using patient-specific cell sources. Currently, the NIDA is funding projects that rely on iPS technologies to: 1) characterize dopaminergic neurotransmission function of neurons derived from iPSCs with genomic aberrations, such as chromosomal polymorphism regions containing dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2) genes; 2) identify genetic variations that can be functionally correlated to addiction in patient cell-derived neurons via iPSCs; 3) make dopamine neurons from patient iPSCs and expose them to methamphetamine and withdrawal in order to analyze the electrophysiological properties of patient-specific neurons versus controls, etc. Even though it is considered a technology with great potential in studies of addiction or other diseases, reprogramming of adult human cells to iPSCs is still an inefficient, tedious and costly process to most researchers. The currently prevailing methods of reprogramming have additional problems such as alteration of genomes or introduction of viruses that can complicate the interpretation of results from the cell-based assays. This project aims to develop a turn-key tool kit that will enable many biomedical researchers to generate iPSCs not only with 10 to 100 folds higher efficiency than other methods, in less than 2 weeks, but also through a feeder-free, xeno-free and footprint-free procedure.