Uninterrupted monitoring of drug effects in human-induced pluripotent stem cell-derived cardiomyocytes with bioluminescence Ca2+ microscopy
Suzuki et al. BMC Res Notes
Uninterrupted monitoring of drug effects in human-induced pluripotent stem cell-derived cardiomyocytes with bioluminescence Ca2+ microscopy
Kazushi Suzuki 0
Takahito Onishi 0
Chieko Nakada 2
Shunsuke Takei 2
Matthew J. Daniels 1
Masahiro Nakano 0
Tomoki Matsuda 0
Takeharu Nagai 0
0 The Institute of Scientific and Industrial Research, Osaka University , 8-1 Mihogaoka, Ibaraki, Osaka 567-0047 , Japan
1 BHF Centre for Regenerative Medicine, Division of Cardiovascular Medicine , West Wing Level 6 , John Radcliffe Hospital, Oxford University , Oxford OX3 9DU , UK
2 NIKON CORPORATION , 471, Nagaodai-cho, Sakae-ku, Yokohama, Kanagawa 244-8533 , Japan
Objective: Cardiomyocytes derived from human-induced pluripotent stem cells are a powerful platform for highthroughput drug screening in vitro. However, current modalities for drug testing, such as electrophysiology and fluorescence imaging have inherent drawbacks. To circumvent these problems, we report the development of a bioluminescent Ca2+ indicator GmNL(Ca2+), and its application in a customized microscope for high-throughput drug screening. Results: GmNL(Ca2+) gives a 140% signal change with Ca2+, and can image drug-induced changes of Ca2+ dynamics in cultured cells. Since bioluminescence requires application of a chemical substrate, which is consumed over ~ 30 min we made a dedicated microscope with automated drug dispensing inside a light-tight box, to control drug addition. To overcome thermal instability of the luminescent substrate, or small molecule, dual climate control enables distinct temperature settings in the drug reservoir and the biological sample. By combining GmNL(Ca2+) with this adaptation, we could image spontaneous Ca2+ transients in cultured cardiomyocytes and phenotype their response to well-known drugs without accessing the sample directly. In addition, the bioluminescent strategy demonstrates minimal perturbation of contractile parameters and long-term observation attributable to lack of phototoxicity and photobleaching. Overall, bioluminescence may enable more accurate drug screening in a high-throughput manner.
hiPSC; Cardiomyocytes; Drug screening; Bioluminescence; Ca2+; Microscope
For decades, a major bottleneck in drug development
has been limited availability of patient-derived tissues
or cells. This changed with the establishment of
humaninduced pluripotent stem cells (hiPSC) [
], which can
now be differentiated into virtually all cell types in vitro.
Patient derived disease-specific hiPSCs recapitulate
many disease phenotypes in culture, which may therefore
serve as a valuable platform for drug discovery or
toxicology testing . This is especially true for the
hiPSCderived cardiomyocytes (hiPSC-CMs) which may enable
a paradigm shift in toxicity testing [
]. However thus far
they have failed to completely recapitulate established
real-world patient based toxicology results in
contemporary platforms [
Drug-induced changes in hiPSC-CMs can be
detected by many methods including classical and
automated electrophysiology or established
fluorescence imaging modalities [
]. However, the inherent
low-throughput of electrophysiological techniques and
physiological perturbation arising from phototoxicity
of fluorescence excitation may introduce limitations
such as recording duration (precluding chronic
toxicity studies), or artefacts.
By contrast biocompatibility of bioluminescence
emission is robustly demonstrated in many eukaryotic
phyla, and as such may provide a better short or long
term imaging solution. Bioluminescent proteins (BPs)
generate detectable emissions by catalyzing a chemical
reaction which consumes a bioluminescent substrate
while releasing a photon in the process. This makes
them totally independent of external light, reducing
phototoxicity. Recent developments of bright BPs such
as Nano-lantern, a chimera of Renilla luciferase (Rluc)
variant and a fluorescent protein (FP), enable
bioluminescence imaging with high signal-to-noise ratio
comparable to fluorescence imaging [
However, performing bioluminescence imaging at scale is
still limited. The faint light associated with
bioluminescence requires placement inside an opaque box in
order to exclude background light. This factor makes
it difficult to test the enormous chemical libraries in a
Here we develop a new bioluminescent Ca2+
indicator GmNL(Ca2+) for hiPSC-CM imaging together with
a customized light-tight box which contains a liquid
dispenser and regional temperature control and can
easily be installed on existing fluorescence
microscopy. Combining GmNL(Ca2+) with the environmental
modifications we demonstrate prolonged visualization
of Ca2+ transients is improved by bioluminescence
compared to fluorescence imaging. Consequently this
strategy in hiPSC-CMs may bring value to drug
development, particularly in chronic toxicology studies
where there is an unmet need.
Materials and methods
Gene construction of GmNL(Ca2+)
General molecular biology experiments were
conducted as described [
]. The sequences of all the
oligonucleotides (Hokkaido System Science,
Hokkaido, Japan) used in this study are provided in
Additional file 1. Each of the cDNAs of C-terminally
deleted Gamillus [
] mutants (ΔC8–11) were
amplified by PCR and digested with BamHI and KpnI. The
digested PCR fragments were cloned in-frame into the
KpnI/EcoRI sites of Nano-lantern(Ca2+)_600/pRSETB
(Addgene#51976) for bacterial expression. To express
the GmNL(Ca2+) in mammalian cells, PCR-amplified
GmNL(Ca2+) genes were inserted into a pcDNA3
mammalian expression vector using BamHI and EcoRI
Protein purification and characterization
Recombinant Gamillus-based NL(Ca2+)_variant proteins
with N-terminal polyhistidine tags were expressed in E.
coli. (JM109(DE3)), purified, and spectroscopically
characterized as described [
Adeno‑associated virus (AAV) production
For the Adeno associated virus expression system,
pHelper and pAAV-DJ were obtained from Cell Biolabs,
Inc. (San Diego, CA, USA). The cDNA of GmNL(Ca2+)
was replaced with ArchT-GFP sequence in
pAAV-CAGArchT-GFP (Addgene#29777). AAV production followed
the manufacturer’s protocol. The titer of AAV vector
was determined by fluorescence titration assay. Briefly,
HEK293T cells were transduced with a range of dilutions
of AAV encoding GmNL(Ca2+). 48 h post transduction,
the percentage of infected cells were assayed with
fluorescence of Gamillus moiety inside GmNL(Ca2+) using
fluorescence microscopy. To ensure a single infection
event per cell, the dilution with less than 40%
fluorescent-positive cells was adopted for calculation of titer.
The virus titer was approximately 1× 108 Infectious units
Customized bioluminescence microscopy
A bespoke assay environment for bioluminescence
imaging was developed for a Ti-E microscope (Nikon
Corporation, Tokyo, Japan) equipped with a x10PlanFluor
(NA 0.3) objective lens and a stage modified for onstage
environmental control (Tokai Hit., Co, Ltd., Shizuoka,
Japan). Drug addition and medium exchange was carried
out using PROcellcare 5030 and PPPump 2010 MiMEDA
enclosed in light-tight box manufactured for this study
(Tokai Hit., Co, Ltd.). Fluorescence and bioluminescence
images were acquired with an EMCCD camera iXon-3
(Andor Technology, Belfast, Northern Ireland) using the
image acquisition software NIS-Elements 4.60 (Nikon
hiPSC‑CM culture and imaging
iCell® Cardiomyocytes (Cellular Dynamics
International, Madison, WI, USA) were purchased, and
aggregates of hiPSC-CMs were prepared as spheroids.
hiPSC-CMs were treated with 10 μl of crude AAV
solution (1 × 106 IFU per aggregates of hiPSC-CMs) for
1–5 weeks before observation. The cells were incubated
at 37 °C, 5% CO2 and culture medium replaced every
3 days. Just before observation hiPSC-CMs were washed
with Tyrodes solution (Sigma-Aldrich, St. Louis, MO,
USA) and exchanged for 20 μM coelenterazine-h (Wako
Pure Chemical Industries, Osaka, Japan) containing
Tyrode solution. For Ca2+ imaging using Fluo4, 5.0 μM
Fluo4-AM (Thermo Fisher Scientific, Waltham, MA,
USA) was loaded onto hiPSC-CMs in Tyrode solution
supplemented with 1xPowerLoad (provided with
Fluo4AM) for 1 h at 37 °C. Fluo4 imaging was conducted with
an FESH0700 IR cut-off filter (Thorlabs, Newton, NJ,
USA) and LED505-C-FL (Nikon Corporation) including
Ex500/20 excitation filter, DM515 dichroic mirror and
EM535/30 emission filter. The images of GmNL(Ca2+)
were acquired with 50 or 30 ms exposure times for
comparison with Fluo4 or drug screening, respectively. For
bioluminescence, 8 × 8 binning was applied to increase
photon counts for each pixel.
For drug studies, the basal Ca2+ transients of
hiPSCCM were recorded, followed by drug incubation for
10 min, and repeated recording of the Ca2+ transient. To
preserve drug activity, they were initially kept at 4 °C on
one side of the chamber and just before use, the
temperature was quickly raised to 37 °C to prevent thermal drift.
The information of all the drugs used in this study are
provided in Additional file 2.
Development and characterization of a bioluminescent Ca2+
In Nano-lantern, the excited energy produced by an
Rluc variant is efficiently transferred to the adjacent FP
by FRET. Since the FP has a higher quantum yield (QY)
the emitted photon number increases. It was possible to
introduce a calcium sensor domain into Nano-lantern
to form Nano-lantern(Ca2+), but this reduced
brightness significantly [
]. To restore brightness we explored
fusions to the recently characterized green FP Gamillus
] which has the highest QY among reported GFPs. We
first swapped Venus from Nano-lantern(Ca2+) with
various C-terminal truncated Gamillus (Δ8–11) constructs
to improve FRET efficiency. The resultant fusion
proteins are designated as Gamillus-based
NL(Ca2+)_variants hereafter (Fig. 1a). Of the tested Gamillus-based
NL(Ca2+)_variants, we found that the Δ9 deletion mutant
exhibited a 140% signal change with comparable
brightness to YNL(Ca2+) when Ca2+-bound (Fig. 1b). Ca2+
titration revealed that the dissociation constant (Kd) for
Ca2+ was 240 nM. To compare the performance of these
Ca2+ indicators to a known standard, we expressed each
Gamillus-based NL(Ca2+)_variant in HeLa cells. Upon
stimulation with histamine, an acute Ca2+ spike
followed by Ca2+ oscillations with smaller amplitudes were
detected with sampling rates up to 10 Hz (Additional
Customized bioluminescence microscopy
Figure 2a shows an inverted microscope customized for
drug screening using bioluminescence imaging. The
system is made of a stage-top incubator, and an automatic
dispenser inside a light-tight box, enabling drug
preparation, drug addition, and medium exchange (Fig. 2b). This
CaM CaM-M13 YYNNLL(Ca2+ )GamCi8llus-Cb9aseCd1N0L(CC1a12+)
Fig. 1 Development of Gamillus based bioluminescent Ca2+ indicators. a Schematic representation of the domain structures of Gamillus-based
NL(Ca2+)_variants, b relative brightness of recombinant Gamillus-based NL(Ca2+)_variants made by deleting 8–11 amino acids at the fusion site
between the FRET acceptor and the split luciferase light donor, with or without Ca2+. Measurements were performed at least in triplicate, and the
averaged data and s.d. are shown
Two 8-well chamber
8-well drug reservoir
incubator itself has two spaces to hold an 8 well chamber
slide for cell observation and eight holes acting as drug
reservoirs (Fig. 2c). Temperature can be controlled
independently for cell observation (25–50 °C) and drug
storage (4–50 °C). Since beating parameters of hiPSC-CMs
exhibit thermal dependence, the temperature inside the
chamber overall should be constant. To validate the
performance of the incubator, Ca2+ transients of hiPSC-CMs
were recorded by Fluo4, a commonly used fluorescent
chemical indicator for Ca2+, at 32, 37 and 42 °C.
Figure 2d shows a typical time course of spontaneous Ca2+
transients at each temperature. The peak-to-peak interval
between Ca2+ transients, commonly employed as a
measure of beat rate, decreased linearly from 2.8 ± 0.29 s at
32 °C to 1.2 ± 0.30 s at 42 °C (Fig. 2e, n = 3).
We then assessed Gamillus-based NL(Ca2+)Δ9
[hereafter GmNL(Ca2+)] signals during spontaneous hiPSC-CM
contraction in the customized microscope. GmNL(Ca2+)
was expressed in cardiomyocyte spheroids by Adeno
associated virus infection (Fig. 3a). GmNL(Ca2+)
was imaged at 20 Hz for 15 min, detecting periodic
changes in bioluminescence signal during synchronized
contractions (Fig. 3b). The time course of
bioluminescence intensity was recorded for the first and last 3 min,
and analyzed to estimate three beating parameters; peak
interval, peak amplitude, and 50% peak width (FWHM)
which approximates to the action potential duration. We
compared these results to those obtained using Fluo4
under various illumination power densities (62, 125,
250 and 500 mW cm−2 respectively). The amplitude of
GmNL(Ca2+) did not change significantly during
observation (− 8.0 ± 21%, n = 8), in contrast to that of Fluo4
which reduced significantly under all illumination
conditions tested (− 50 ± 8.5% at 62 mW cm−2 to − 99 ± 0.51%
at 500 mW cm−2) indicative of dye loss by export or
photobleaching (Fig. 3c).
In addition, stability of beating parameters between
the start and the end of the observation window was only
seen with GmNL(Ca2+) (From 1.3 ± 0.36 to 1.3 ± 0.27 s
for beat–beat interval, and 0.61 ± 0.14 to 0.59 ± 0.063 s
for FWHM, n = 8) and Fluo4 at the lowest power density
(62 mW cm−2) (from 1.6 ± 0.11 to 1.8 ± 0.10 s for
interval, and from 0.48 ± 0.029 to 0.59 ± 0.031 s for FWHM,
n = 6). At higher power density reduction in
peak-topeak interval (From 1.9 ± 0.10 to 0.99 ± 0.051 s at 500
mW cm−2, n = 7) was seen suggesting extrinsic light
Fig. 3 Ca2+ imaging with GmNL(Ca2+) in hiPSC-CMs. a
Bioluminescence and bright-field images of hiPSC-CMs expressing
GmNL(Ca2+). Scale bar, 50 μm. b Representative time course of the
signal of GmNL(Ca2+), and Fluo4 at 0 or 15 min. Images were taken
at 20 Hz, 20 μM coelenterazine-h was added just before imaging.
Excitation light at 62 mW cm−2 for Fluo4 was applied continuously
for 15 min. The measurements were replicated (n ≥ 6) for each
condition. c Relative change in beating parameters after 15 min
continuous imaging. Data are presented as mean ± S.D.; n = 6–8.
Ca2+ imaging with Fluo4 was conducted under four different
power densities of excitation light, 62, 125, 250 and 500 mW cm−2
respectively. d Representative time course of the bioluminescence
signal of GmNL(Ca2+) before and after treatment with either 1 μM
isoproterenol or 40 μM propranolol. The measurements were
triplicated for each drug. e Mean beat interval before and after
treatment; data are presented as mean ± S.D. Two-tailed Student’s t
test was performed. **p < 0.01; n = 3
illumination can cause significant phototoxicity in this
system (Fig. 3c). Collectively, these observations
demonstrate that GmNL(Ca2+) improves long-term signal
stability without physiological perturbations arising from
extrinsic illumination. Interestingly the variation between
hiPSC-CMs from GmNL(Ca2+) was greater than that
from cells labelled with Fluo4.
Drug‑induced changes to the hiPS‑CM Ca2+ transient
Next we tested whether GmNL(Ca2+) can identify
expected Ca2+ transient changes in hiPSC-CMs induced
by well characterized drugs. After addition of
isoproterenol, a non-selective β-adrenergic agonist used clinically
to increase the heart rate, the mean peak-to-peak interval
reduced (from 1.1 ± 0.16 to 0.75 ± 0.070 s, n = 3) (Fig. 3d,
]. Conversely, propranolol, an adrenergic
receptor blocker, increased the peak-to-peak interval (from
1.0 ± 0.081 to 1.27 ± 0.11 s, n = 3) as expected (Fig. 3d, e)
]. Similarly GmNL(Ca2+) could correctly elicit the rate
related Ca2+ transient alterations induced by Dopamine,
and Doxazosin (Additional file 4). These results suggested
that GmNL(Ca2+) is able to report bidirectional drug
effects in hiPSC-CMs using a drug dispensing system
based inside the on-stage environmental control
conditions needed for bioluminescence imaging.
GmNL(Ca2+) enables imaging free from the problems
of phototoxicity and photobleaching, which plague
fluorescence imaging. In contrast to our
expectations, GmNL(Ca2+) was slightly dimmer than
Nanolantern(Ca2+) at saturating Ca2+ concentrations.
Saturation mutagenesis at the junction between the light
donor and the FRET acceptor might improve the
performance of GmNL(Ca2+). Although the GmNL(Ca2+)
measurements in hiPSC-CM show stable Ca2+ transients
between the beginning and the end of the observation
window, the variation between hiPSC-CMs appears high
in comparison with that from Fluo4. The observed
variability might be attributable to heterogeneous infection of
AAV or reduced penetration of the luminescent substrate
into the spheroid culture model as either may lower
The spontaneous beating characteristics of
cardiomyocytes are sensitive to the physical environment, especially
temperature, therefore maintenance of sample
environment is crucial. In our system we can independently
control the temperature of the sample chamber and the drug
reservoir, remotely adding the small molecules without
perturbation of imaging environment.
Overall, our study presents a bioluminescent Ca2+
indicator and a light-tight box equipped with an
automatic dispenser that can be controlled remotely. As a
proof-of-concept, we demonstrate a minimally
harmful, and operator independent Ca2+ imaging strategy in
hiPSC-CMs with robust testing of drug-induced changes
in Ca2+ transients at a scale.
As GmNL(Ca2+) is intensiometric indicator, the
oscillation from GmNL(Ca2+) in hiPSC-CMs should include the
fraction of motion artefact in addition to Ca2+-dependent
signal as previously shown [
]. A negative control using
Ca2+-insensitive probe will give the insightful
information about motion artefact.
Additional file 1. Oligonucleotides used in this study.
Additional file 2. Drugs used in this study.
Additional file 3. Characterization of the bioluminescent Ca2+ indicators
in HeLa. (a) A series of pseudo-coloured ratio images of HeLa cells
expressing GmNL(Ca2+), following 10 μM histamine stimulation (arrow). Scale bar,
10 μm. (b) Time course of the B/B0 ratio change at an ROI (white box in (a)).
Number indicates the time point of each image in (a).
Additional file 4. Evaluation of mean beat interval before and after
treatment with either 10 μM Dopamine or 10 μM Doxazosin. Two-tailed Student’s
t-test was performed. **p<0.01; Data are presented as mean±S.D.; n=3.
hiPSC: human-induced pluripotent stem cells; CM: cardiomyocytes; BP:
bioluminescent protein; GFP: green fluorescent protein; AAV: adeno-associated
virus; QY: quantum yield; FWHM: full width at half maximum; Rluc: Renilla
luciferase; FRET: Förster resonance energy transfer.
KS and TO contributed equally to this work. TN conceived and coordinated the
project; KS, TO, MN, TM, CN and ST designed the experiments; CN and ST
optimized the microscopy; KS and TO constructed and characterized GNL(Ca2+)
in vitro, and performed the Ca2+ imaging of hiPSC-CM with support of CN
and ST; TO assayed the drug-induced alteration of Ca2+ transient in hiPSC-CM
with support of CN, ST and MJD; all authors analyzed data; KS, TO, CN, TM and
TN wrote the paper, with contributions from all authors. All authors read and
approved the final manuscript.
The authors wish to thank Dr. Yamada from Nikon cooperation for their help
in preparation of hiPSC-CM. pAAV-CAG-ArchT-GFP (Addgene plasmid # 29777)
was a gift from Edward Boyden.
The authors declare that they have no competing interests.
Availability of data and materials
The data that support the findings of this study are available from the
corresponding author on request. The nucleotide sequences of Gamillus-based
NL(Ca2+)_variants have been deposited to DDBJ database under the following
entry IDs: LC325493 (Gamillus-based NL(Ca2+)_ΔC8), LC325494 (Gamillus-based
NL(Ca2+)_ΔC9), LC325495 (Gamillus-based NL(Ca2+)_ΔC10) and LC325496
(Gamillus-based NL(Ca2+)_ΔC11), respectively.
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