Optogenetic Multiparametric Assay for HT Cardiotoxicity Testing

描述(由申请人提供)：高通量监测单个心肌细胞的生理学尚未见报道。无法进行高通量的生理测量限制了许多基础和应用研究，包括在心脏毒性测试中使用干细胞来源的心肌细胞。目前的自动化心脏毒性测试预测价值很低，因为它们使用的是由单通道设计的肿瘤细胞系(例如HERG)，而且生理相关测试仅为少数候选细胞在相对较晚的开发阶段保留。这些模型的生物学相关性很差，导致候选药物在FDA批准之前甚至在商业化之后的失败率很高。我们已经从心肌细胞自动记录了钙瞬变，但仍然受到电极起搏设备的限制，这种起搏设备阻止了超过96孔格式的小型化。此外，作为可兴奋细胞中最相关的生理参数，动作电位测量仍然保留在低通量分析中。我们提出了一些解决这些问题的概念上的进展，通过开发一种小型化的、基于细胞的光基因起搏设备来高通量分析来自人诱导多能干细胞(HiPSC)的心肌细胞，在一个用于心肌细胞生理的逐细胞细胞术分析的自动化平台中。我们还将通过荧光电压探针和录音后跟踪来开发动作电位(AP)的自动分割/分析，以在固定和免疫染色分析后识别相同的细胞。已经在以前的SBIR合同中开发的钙瞬变(CT)分析将与AP和记录后跟踪相结合，以生成以高吞吐量进行的所有这些终端的单细胞多参数测量。将通过不同的机制对改变AP的药物进行广泛的评估，以验证该平台。初步数据显示，表达光触发蛋白通道视紫红质-2(ChR2)的稳定细胞株将与心肌细胞电耦合，允许在不破坏正常心肌细胞生理的情况下对AP进行光学控制刺激。膜AP可以通过电压探针记录在心肌细胞中，适合于图像分割分析。自动CT测量和hPSC来源的心肌细胞是检测参比药物心脏毒性效应的有效模型。这些目标将推动使用荧光探针来测量动作电位、钙离子通量和细胞对刺激的反应特性。心肌细胞生理学将通过图像分析软件进行量化，该软件记录和分析单细胞AP和CT与心脏亚型或特定蛋白表达的关系。该软件将把图像分割成单个细胞的记录，因此所有的测量和数据分析都将以细胞为基础。将对384井和1536井的格式进行评估，以对每个单独数据点进行数百个细胞的筛选(例如，化合物测试)，允许在资助期结束前在一个屏幕上处理数万到数十万个数据点。将对通道开放器和阻断器进行测试，以验证平台的有效性。该平台将在基础和应用研究中找到应用，包括再生医学研究和药物开发/安全测试。

DESCRIPTION (provided by applicant): Monitoring physiology of individual cardiomyocytes in high throughput has not been reported. The inability to perform high throughput physiological measurements limits many basic and applied studies, including the use of stem cell derived cardiomycoytes in cardiotoxicity testing. Current automated cardiotoxicity tests have poor predictive value because they use tumor cell lines engineered with single channels (e.g. hERG), and physiologically relevant tests are reserved for few candidates during the relatively late stages of development. The poor biological relevance of these models contributes to the high failure rate of drug candidates before FDA approval and even after commercialization. We have automated recording from myocytes for Calcium Transients, but are still limited by use of electrode devices for pacing that prevents miniaturization beyond 96- well format. Furthermore, Action Potential measurement, the most relevant physiological parameter in excitable cells, is still reserved to low throughput analysis. We propose several conceptual advances to solve these problems by developing a miniaturized, cell-based optogenetic pacing device for high throughput analysis of human Induced Pluripotent Stem Cell (hIPSC)-derived cardiomyocytes in an automated platform for cell-by-cell cytometric analysis of cardiomyocyte physiology. We will also develop automatic segmentation/analysis of Action Potentials (AP) through fluorescent voltage probes and post-recording tracking to identify the same cells after fixation and immunostaining analysis. Calcium Transient (CT) analysis, already developed in a previous SBIR contract, will converge with AP and post-recording tracking to generate single cell multiparametric measurement of all these endpoints conducted in High Throughput. Extensive evaluation will be conducted with drugs that alter AP through different mechanisms to validate the platform. Preliminary data show that stable cell lines expressing the light-triggered protein Channelrhodopsin-2 (ChR2) will electrically couple to cardiomyocytes, allowing optically controlled stimulation of AP without disruption of normal cardiomyocyte physiology. Membrane AP can be recorded in cardiomyocytes through voltage probes and are suitable to image segmentation analysis. Automatic CT measurement and hIPSC-derived cardiomyocytes are an effective model to test cardiotoxic effects of reference drugs. The Aims will advance the use of fluorescent probes to measure action potential, calcium flux and cell characteristics in response to the stimulation. Cardiomyocyte physiology will be quantified by image analysis software that records and analyzes single-cell AP and CT in relation to cardiac subtype or specific protein expression. The software will segment the images into single cell recordings, thus all measurements and data analysis will be on a cell-by- cell basis. The format will be evaluated for 384- and 1536-well to conduct screening on hundreds of cells per individual data point (e.g. compound tested), allowing throughput of tens to hundreds of thousands of datapoints in a single screen by the end of the funding period. Channel openers and blockers will be tested to validate the platform. The platform will find applications in basic and applied research, including regenerative medicine research and drug development/safety testing.