A novel “optical dynamic clamp” method to make iPSC-CMs a more viable platform for drug screening

一种新颖的“光学动态钳”方法使 iPSC-CM 成为更可行的药物筛选平台

• 批准号: 9353192
• 负责人: Bonnie Quach
• 金额: $ 4.4万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9353192
• 关键词: Action Potentials; Adult; Anti-Arrhythmia Agents; Back; Basic Science; Behavior; Biological Assay; Cardiac Myocytes; Cardiotoxicity; Cells; Characteristics; Clinical; Closure by clamp; Coupled; Dependence; Development; Disease; Electrophysiology (science); Ensure; Feedback; Generations; Human Activities; Individual; Injectable; Investigation; Ion Channel; Kinetics; Measures; Membrane Potentials; Methods; Morphology; Nature; Optics; Patients; Pharmaceutical Preparations; Phenotype; Physiological; Potassium; Preclinical Drug Evaluation; Proton Pump; Rest; Role; Sensitivity and Specificity; System; Techniques; Therapeutic; Time; Tissues; base; clinically relevant; design; drug development; drug discovery; drug mechanism; drug use screening; dynamic system; effective therapy; experimental study; fetal; improved; in vitro Model; in vivo; induced pluripotent stem cell; novel; optogenetics; patch clamp; patient population; prevent; research and development; screening; tool; voltage; voltage sensitive dye

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have the potential to provide a relevant platform for basic research and drug development. The advantages of these cells over current in vitro models used to investigate the effect of a drug on electrical activity are that they are of human origin and can be derived directly from the target patient population. However, one major obstacle to using iPSC-CMs is that they possess an immature phenotype, making it difficult to determine if resulting behavior reflects adult cardiomyocyte behavior. It has been demonstrated that a missing inward rectifier potassium current, IK1, can be artificially re-introduced via “dynamic clamp”, an electrophysiological technique that allows for control of electrical input based on real-time feedback and analysis of membrane potential. This approach makes iPSC-CMs more electrophysiologically adult-like, enabling the investigation of the effect of drugs on action potential morphology and kinetics. However, this method is tedious, low throughput, and can only be performed on single cells. We aim to improve upon this approach by applying optogenetic techniques in combination with optical mapping in iPSC-CM beating clusters to create a novel “optical dynamic clamp” platform for screening. Key points:  The design of the optical dynamic clamp will be based on the same principles of traditional dynamic clamp.  ArchT, a hyperpolarizing optogenetic proton pump, will be used to artificially compensate for the missing IK1 component in the beating clusters of iPSC-CMs and push its electrical maturity.  Optical mapping with voltage sensitive dyes will be used to extract information on action potential morphology and kinetics as well as provide the necessary real-time information to control the dynamic system.  This platform does not restrict us to using only single cells, but allows for the use of beating clusters of iPSC-CMs. Beating clusters offers the advantage of being more tissue-like since individual cardiomyocytes will be electrically coupled to its neighbors, similarly to in vivo conditions. Importantly, the optical nature of the approach (in contrast to patch clamping) will enable high- throughput drug screening using iPSC-CMs in a more tissue-like format of cells, allowing for more meaningful interpretations of disease and drug mechanisms of action.

诱导性多能干细胞衍生的心肌细胞（iPSC-CM）具有提供 基础研究和药物开发的相关平台。这些电池相对于现有电池的优点 用于研究药物对电活动的影响的体外模型是 来源，可以直接从目标患者人群中获得。然而，一个主要障碍是， 使用iPSC-CM的另一个缺点是它们具有不成熟的表型，这使得难以确定是否产生 行为反映了成年心肌细胞的行为。已经证明，缺失的内向整流器 钾电流IK 1可以通过“动态钳”人工重新引入， 允许基于实时反馈和分析来控制电输入的技术 膜电位这种方法使iPSC-CM在电生理上更像成人， 研究药物对动作电位形态学和动力学的影响。但这 该方法繁琐、低通量，并且只能在单个细胞上进行。我们的目标是改善 通过将光遗传学技术与光学作图相结合， iPSC-CM跳动集群创建一个新的“光学动态钳”筛选平台。 关键点： 光学动态夹的设计将基于与传统光学夹相同的原理。 动态箝位 将使用超极化光遗传质子泵NARCHT来人工补偿 在iPSC-CM的跳动簇中缺失IK 1组分并推动其电成熟。 将使用电压敏感染料的光学映射来提取动作电位信息 形态学和动力学以及提供必要的实时信息来控制 动态系统 这个平台并不限制我们只能使用单个细胞，而是允许使用跳动 iPSC-CM簇。搏动簇提供了更像组织的优点，因为 单个心肌细胞将与其相邻细胞电偶联，类似于体内 条件 重要的是，该方法的光学性质（与膜片钳相反）将使高- 在更像组织的细胞形式中使用iPSC-CM进行通量药物筛选， 对疾病和药物作用机制的有意义的解释。