Revolutionary Technology for Efficient Derivation of Human iPSCs with Messenger R

描述(由申请人提供)：该项目的目标是开发一种商业试剂盒，使正在进行基于细胞模型的项目的药物成瘾研究人员能够成功和方便地生产人类诱导多能干细胞(IPSCs)。吸毒成瘾摧毁了吸毒者及其家人的生活，造成了巨大的社会问题，并给国家的医疗保健系统带来了成本。导致成瘾的分子机制在很大程度上是未知的，部分原因是缺乏患者的神经细胞类型。使人类ipscs成为可能的技术打开了通过使用患者特定的细胞来源直接询问成瘾的病理生理学的新领域的大门。目前，NIDA正在资助依赖iPS技术的项目：1)表征具有基因组异常的ipSCs来源的神经元的多巴胺能神经传递功能，例如含有多巴胺转运体(DAT)和囊泡单胺转运体(VMAT2)基因的染色体多态区；2)通过IPSCs识别在功能上与患者细胞来源的神经元成瘾相关的遗传变异；3)从患者的IPSCs中制造多巴胺神经元，并将其暴露于甲基苯丙胺和戒断，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IPSCs仍然是一个低效、繁琐和昂贵的过程。目前流行的重新编程方法还有其他问题，如基因组的改变或病毒的引入，这可能会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不仅能够在不到2周的时间内以比其他方法高10到100倍的效率产生IPSCs，而且还可以通过一种无饲养器、无异种和无足迹的程序来产生IPSCs。

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.