High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

被引:0
|
作者
Chang, Yu-Fen [1 ]
Su, Wan-Chi [1 ]
Su, Chih-Chuan [1 ]
Chung, Min-Wen [1 ]
Chang, Jin [2 ]
Li, You-Yi [3 ]
Kao, Yi-Ju [3 ]
Chen, Wen-Pin [3 ]
Daniels, Matthew J. [4 ,5 ,6 ]
机构
[1] LumiSTAR Biotechnol Inc, Taipei, Taiwan
[2] NEXEL Co Ltd, Seoul, South Korea
[3] Natl Taiwan Univ, Coll Med, Inst Pharmacol, Taipei, Taiwan
[4] Manchester Univ NHS Fdn Trust, Manchester Royal Infirm, Manchester Heart Ctr, Manchester, Lancs, England
[5] Univ Manchester, Manchester Acad Hlth Sci Ctr, Div Cardiovasc Sci, Manchester, Lancs, England
[6] Univ Manchester, Div Cell Matrix Biol & Regenerat Med, Manchester, Lancs, England
来源
关键词
STEM-CELLS; EXPRESSION; INDICATORS; PROMOTER;
D O I
10.3791/63175
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cellderived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.
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页数:15
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