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报道。无法进行高通量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衍生的心肌细胞。目前的自动化心脏毒性测试的预测价值较差，因为它们使用的是单通道工程的肿瘤细胞系（例如hERG），并且在相对较晚的发展阶段，生理学相关的测试保留给少数候选细胞。这些模型的生物学相关性较差，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。我们已经从肌细胞中自动记录钙瞬态，但仍然受到电极起搏装置使用的限制，防止超过96孔格式的小型化。此外，动作电位测量作为可兴奋细胞中最相关的生理参数，仍然保留在低通量分析中。为了解决这些问题，我们提出了几个概念上的进展，通过开发一种小型化的、基于细胞的光遗传学起搏装置，用于在一个自动化平台上对人类诱导多能干细胞（hIPSC）衍生的心肌细胞进行高通量分析，从而对心肌细胞生理学进行逐细胞细胞分析。我们还将开发动作电位（AP）的自动分割/分析，通过荧光电压探针和记录后跟踪来识别固定和免疫染色分析后的相同细胞。在之前的SBIR合同中已经开发的钙瞬态（CT）分析，将与AP和记录后跟踪融合，在高通量下生成所有端点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）的稳定细胞系将与心肌细胞电偶联，允许光控制AP刺激而不破坏正常的心肌细胞生理。通过电压探针可以记录心肌细胞的膜AP，适合于图像分割分析。自动CT测量和hipsc来源的心肌细胞是检测参比药物心脏毒性作用的有效模型。Aims将推进荧光探针的使用，以测量动作电位，钙通量和细胞特性响应刺激。心肌细胞生理学将通过图像分析软件进行量化，该软件记录和分析与心脏亚型或特定蛋白质表达相关的单细胞AP和CT。该软件将图像分割成单个细胞记录，因此所有的测量和数据分析将以细胞为基础。该格式将在384井和1536井中进行评估，以便在每个数据点（例如化合物测试）上对数百个细胞进行筛选，从而在资助期结束时在单个筛选中实现数万至数十万个数据点的吞吐量。将测试通道开启器和阻塞器以验证该平台。该平台将应用于基础研究和应用研究，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.