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. 2015 Oct 13;5(4):582-96.
doi: 10.1016/j.stemcr.2015.08.009. Epub 2015 Sep 12.

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

Rami Shinnawi  1 , Irit Huber  1 , Leonid Maizels  1 , Naim Shaheen  1 , Amira Gepstein  1 , Gil Arbel  1 , Anke J Tijsen  1 , Lior Gepstein  2
Affiliations

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

Rami Shinnawi et al. Stem Cell Reports. .

Abstract

The advent of the human-induced pluripotent stem cell (hiPSC) technology has transformed biomedical research, providing new tools for human disease modeling, drug development, and regenerative medicine. To fulfill its unique potential in the cardiovascular field, efficient methods should be developed for high-resolution, large-scale, long-term, and serial functional cellular phenotyping of hiPSC-derived cardiomyocytes (hiPSC-CMs). To achieve this goal, we combined the hiPSC technology with genetically encoded voltage (ArcLight) and calcium (GCaMP5G) fluorescent indicators. Expression of ArcLight and GCaMP5G in hiPSC-CMs permitted to reliably follow changes in transmembrane potential and intracellular calcium levels, respectively. This allowed monitoring short- and long-term changes in action-potential and calcium-handling properties and the development of arrhythmias in response to several pharmaceutical agents and in hiPSC-CMs derived from patients with different inherited arrhythmogenic syndromes. Combining genetically encoded fluorescent reporters with hiPSC-CMs may bring a unique value to the study of inherited disorders, developmental biology, and drug development and testing.

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Figures

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Graphical abstract
Figure 1
Figure 1
ArcLight Expression and Imaging (A) Lentiviral transduction of the hiPSC-CMs led to robust ArcLight expression, manifested by cyclic changes in the cell fluorescence with reduced intensity during depolarization. Scale bars, 20 μm. (B and C) Optical APs derived from line-scan imaging of ArcLight-expressing hiPSC-CMs. (D) Optical APs revealing ventricular-, atrial-, and nodal-like AP morphologies. (E–G) Changes in AP morphologies (top-left images, red signals depict post-treatment tracings), following the application of blockers of the fast (E4031, 500 nM, n = 27 in three independent experiments, E) and slow (Chromanol293B, 30 μM, n = 29 in three independent experiments, F) components of the delayed rectifier potassium currents and activator of the late-sodium current (ATX-II-30nM, n = 28 in three independent experiments, G). All agents caused significant APD90 prolongation (right) and development of EADs and triggered beats (lower). ∗p < 0.01 when compared to baseline values. (H) No significant differences were noted after control-solution application (DMSO 0.1% v/v, n = 16 in three independent experiments). Error bars represent SEM.
Figure 2
Figure 2
Establishment of Stable Transgenic ArcLight-hiPSCs (A) Expression of ArcLight (green fluorescence) by undifferentiated ArcLight-hiPSC colonies (top), but not by the parental hiPSC colonies (bottom). The merged light (DIC) and fluorescent images (right) are also shown. Scale bars, 50 μm. (B) Immunostaining of ArcLight-hiPSC colonies for OCT4, NANOG, SSEA4, and TRA-1-60. (C) Immunostaining of ArcLight-hiPSC-CMs for sarcomeric α-actinin and cTnI. (D) qPCR analysis of ArcLight-hiPSC-CMs showing expression of cardiac-specific genes (NKX2.5, MLC-2V, MYH-6, and MYH-7) and downregulation of pluripotent genes (OCT4 and NANOG). Experiments included three biological replicates measured as two technical replicates. (E and F) ArcLight expression by hiPSC-CMs monolayers (E) and single-dispersed cells (F) and the derived optical AP. Note the increase in fluorescence during repolarization (resting state) and the decreased intensity during depolarization (AP development). Scale bar, 20 μm. (G) Optical tracings analysis used to measure APD90. This process can be scaled up to analyze several cells (bottom). Error bars represent SEM. See also Figure S2.
Figure 3
Figure 3
Simultaneous ArcLight and di-8-ANEPPS Recordings from hiPSC-CMs (A) Short-term recordings revealing stable optical APs over a 1 hr period (left). Note also the stable APD90 values (right, n = 14 in three independent experiments). See also Figure S3A. (B) Repeated phenotyping of the same cell over several days. (C) Optical APs acquired simultaneously using ArcLight (black tracing) and di-8-ANEPPS (red tracing). (D) High correlation (R2 = 0.98) between ArcLight- and di-8-ANNEPS-based APD90 measurements from the same cells. (E) Improved signal-to-noise ratio (SNR) values in the ArcLight optical signals when compared to di-8-ANNEPS analysis (∗p < 0.001, n = 17 in three independent experiments). (F) Changes in the optical-signal intensity at 15 and 30 s of continuous imaging. Note the significant attenuation of the di-8-ANEPPS signal (due to photobleaching) in contrast to the limited effect on the ArcLight signal (∗p < 0.001, n = 17 in three independent experiments). (G) Optical APs recorded from ArcLight-hiPSC-CMs during pacing at different rates. (H) Restitution plot depicting the effects of the pacing cycle-length on APD90 values (n = 7 in three independent experiments). Note the typical shortening of APD90 values at faster rates. Error bars represent SEM.
Figure 4
Figure 4
Drug Testing Using ArcLight-hiPSC-CMs (A–C) Evaluating the effects of different hERG (IKr) blockers, including cisapride (100 nM, n = 15 in three independent experiments, A), erythromycin (30 μM, n = 29 in three independent experiments, B), and sotalol (20 μM, n = 22 in three independent experiments, C) on the optical APs of ArcLight-hiPSC-CMs. Note the resulting AP prolongations (red tracings, left), leading to significant increases in APD90 values (middle) and the development of EADs in several (27%, 12%, and 36%, respectively) hiPSC-CMs (right). ∗p < 0.01 when compared to baseline recordings. (D) AP prolongation (left and middle) and arrhythmogenic activity (right) in ArcLight-hiPSC-CMs, following 1 hr treatment with the TKI nilotinib (1 μM, n = 17 in three independent experiments, ∗p < 0.01). Error bars represent SEM.
Figure 5
Figure 5
ArcLight Imaging of LQTS2 hiPSC-CMs (A) Differentiation of the LQTS2-hiPSCs (representative colony shown on the left; scale bar, 100 μm) into cardiomyocytes, positively stained for sarcomeric α-actinin (middle) and cTnI (right). Scale bars, 20 μm. (B) ArcLight imaging showing prolonged optical APs in the LQTS2-hiPSC-CMs (bottom) when compared to healthy control cells (top). (C) Summary of the average APD90 values, measured at different beating rates, in the healthy control (blue, n = 203 in 19 independent experiments) and LQTS2 (red, n = 97 in 11 independent experiments) hiPSC-CMs. Note the significantly longer (∗p < 0.01) APD90 values in the LQTS2-hiPSC-CMs at all rates, but especially at slow beating rates. (D) Typical recordings from the LQTS2-hiPSC-CMs showing the development of EADs, triggered beats, and sustained arrhythmogenic activity. Error bars represent SEM.
Figure 6
Figure 6
GCaMP5G Expression and Calcium Imaging of hiPSC-CMs (A) Lentiviral transduction of hiPSC-CMs led to robust GCaMP5G expression and cyclic changes in the cell fluorescence with an increase in intensity concomitant with the rise in intracellular calcium concentration. Scale bars, 20 μm. (B) Line-scan imaging (using the red line in A) showing typical optical intracellular calcium transients. (C–E) Optical calcium transients (C) acquired simultaneously from the same cells (n = 16 in three independent experiments) using GCaMP5G (black tracing) and Rhod-3 (red tracing). The transients displayed similar general appearance and decay times, while the rise time was slightly longer in the GCaMP5G signal (∗p < 0.01) (D). SNR values were significantly higher (∗p < 0.001) for the GCaMP5G optical tracings when compared to the Rhod-3 signals (E). (F) Continuous optical calcium recordings from the GCaMP5G-hiPSC-CMs (top). The GCaMP5G signals displayed higher mean amplitude (lower left) and significantly less attenuation in fluorescent intensity over time when compared to Rhod-3 signals. ∗p < 0.01. (G) Drug testing using GCaMP5G-hiPSC-CMs. Isoproterenol (1 μM) led to a significant increase in the beating rate (shorter cycle-length), as well as a shorter decay and rise times of the calcium transient (∗p < 0.05, ∗∗p < 0.01, n = 21 in three independent experiments). (H) Application of ouabain led to a dose-dependent arrhythmogenic activity, ranging from local calcium release events, double- and triple-humped transients, to sustained triggered activity. (I) ArcLight-hiPSC-CMs were loaded with Rhod-3. Combined line-scan imaging allowed simultaneous recording of both the optical AP (black) and intracellular calcium transient (red). (J) Combined voltage (black tracing) and calcium (red) imaging using hiPSC-CMs transduced to express both Arch(D95N) and GCaMP5G. Error bars represent SEM. See also Figure S3B.
Figure 7
Figure 7
GCaMP5G-Based Calcium Imaging of CPVT2-hiPSC-CMs (A) Differentiation of the CPVT2-hiPSCs (left; scale bar, 100 μm) into cardiomyocytes positively stained for sarcomeric α-actinin (middle) and cTnI (right). Scale bars, 20 μm. See also Figure S4. (B) qPCR analysis of the CPVT2-hiPSC-CMs showing expression of cardiac-specific genes (NKX2.5, MLC-2V, MYH-6, and MYH-7) and downregulation of OCT4 and NANOG. Experiments included three biological replicates measured as two technical replicates. (C and D) GCaMP5G optical calcium imaging of healthy control (C) and CPVT2 (D) hiPSC-CMs. The CPVT2-cardiomyocytes were highly arrhythmogenic, displaying diastolic local calcium release events (top), double-humped signals (second panel), and sustained arrhythmias (bottom). (E) An example of new arrhythmias in a CPVT2-hiPSC-CM (bottom) caused by isoproterenol. Error bars represent SEM.
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