Monitoring Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes with Genetically Encoded Calcium and Voltage Fluorescent Reporters

摘要

class="head no_bottom_margin" id="sec1title">IntroductionThe ability to reprogram adult somatic cells into pluripotent stem cells by a set of transcription factors has revolutionized biomedical research (, ). The generated human-induced pluripotent stem cells (hiPSCs) can be coaxed to differentiate into a variety of cell lineages (including cardiomyocytes [, ]) that can then be utilized for the development of autologous cell-replacement therapies, disease modeling, and drug discovery ().In the cardiac field, hiPSC lines were established from healthy individuals (, ) and from patients inflicted with acquired (heart failure) () and inherited cardiac disorders. Among the latter, patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs) models of different inherited arrhythmogenic syndromes (, , , , , ) and diverse cardiomyopathies (, href="#bib29" rid="bib29" class=" bibr popnode">Sun et al., 2012) were established. The patient/disease-specific hiPSC-CMs were shown to recapitulate the disease phenotypes in culture, to provide mechanistic insights into disease processes, and to evaluate existing and novel therapies. Similarly, hiPSC-CMs were also proposed as a valuable tool for drug development (href="#bib24" rid="bib24" class=" bibr popnode">Mercola et al., 2013), demonstrating, for example, their value for safety pharmacology by screening the proarrhythmic effects of certain compounds (href="#bib5" rid="bib5" class=" bibr popnode">Braam et al., 2013, href="#bib21" rid="bib21" class=" bibr popnode">Liang et al., 2013, href="#bib35" rid="bib35" class=" bibr popnode">Zwi et al., 2009).One of the key prerequisites for achieving the goals of these applications is to develop efficient tools to study the functional properties of the hiPSC-CMs and specifically of their electrophysiological and excitation-contraction-coupling properties. To this end, different electrophysiological techniques (patch-clamp (href="#bib10" rid="bib10" class=" bibr popnode">Itzhaki et al., 2011a) and multielectrode extracellular potential recordings [href="#bib35" rid="bib35" class=" bibr popnode">Zwi et al., 2009]) and imaging modalities (using voltage- or calcium-sensitive fluorescent dyes) were utilized. While providing valuable information, these methodologies also display inherent limitations, such as relatively low-throughput (patch-clamp), limited electrophysiological information (extracellular recordings), phototoxicity (voltage and calcium sensitive dyes), and inability to obtain long-term repeated recordings (patch-clamp, fluorescent dyes). Consequentially, a method that allows long-term, serial, and cellular functional phenotyping of healthy and diseased hiPSC-CMs is direly needed, especially if it can be achieved in a non-invasive, high-resolution, and large-scale manner.The developments in the field of genetically encoded fluorescent indicators may provide a possible solution to the aforementioned challenges. Genetically encoded indicators are composed of a sensing element, which is usually fused to an autofluorescent protein (like circularly permuted enhanced GFP; cpEGFP) that alters its fluorescent intensity as a result of conformational changes in the sensing element. While utilized in numerous neuroscience-related experimental models (href="#bib2" rid="bib2" class=" bibr popnode">Akemann et al., 2010, href="#bib6" rid="bib6" class=" bibr popnode">Cao et al., 2013, href="#bib9" rid="bib9" class=" bibr popnode">Grienberger and Konnerth, 2012, href="#bib22" rid="bib22" class=" bibr popnode">Looger and Griesbeck, 2012, href="#bib32" rid="bib32" class=" bibr popnode">Tian et al., 2009), the use of similar indicators in non-neuronal tissues, such as the heart, has been more limited (href="#bib1" rid="bib1" class=" bibr popnode">Addis et al., 2013, href="#bib8" rid="bib8" class=" bibr popnode">Chong et al., 2014, href="#bib15" rid="bib15" class=" bibr popnode">Kaestner et al., 2014, href="#bib20" rid="bib20" class=" bibr popnode">Leyton-Mange et al., 2014). Here, we aimed to transfer these emerging technologies to the cardiac field, specifically focusing on genetically encoded calcium indicators (GECIs) (href="#bib9" rid="bib9" class=" bibr popnode">Grienberger and Konnerth, 2012, href="#bib15" rid="bib15" class=" bibr popnode">Kaestner et al., 2014, href="#bib32" rid="bib32" class=" bibr popnode">Tian et al., 2009) and genetically encoded voltage indicators (GEVIs) (href="#bib13" rid="bib13" class=" bibr popnode">Jin et al., 2012, href="#bib17" rid="bib17" class=" bibr popnode">Kralj et al., 2012, href="#bib20" rid="bib20" class=" bibr popnode">Leyton-Mange et al., 2014), in an attempt to establish experimental platforms to monitor the functional activity of hiPSC-CMs. To this end, we aimed to express GCaMP5G (href="#bib1" rid="bib1" class=" bibr popnode">Addis et al., 2013, href="#bib32" rid="bib32" class=" bibr popnode">Tian et al., 2009), a GECI that displays improved dynamic range, improved sensitivity, and maintains relatively stable expression levels, and ArcLight A242 (href="#bib6" rid="bib6" class=" bibr popnode">Cao et al., 2013, href="#bib13" rid="bib13" class=" bibr popnode">Jin et al., 2012, href="#bib20" rid="bib20" class=" bibr popnode">Leyton-Mange et al., 2014), a new variant of the Ciona intestinalis voltage-sensitive (CiVS)-based fluorescent protein voltage sensor (href="#bib3" rid="bib3" class=" bibr popnode">Barnett et al., 2012, href="#bib26" rid="bib26" class=" bibr popnode">Murata et al., 2005) super-family, in both healthy and diseased hiPSC-CMs.