Biowire: A platform for maturation of human pluripotent stem cell-derived cardiomyocytes

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DOI: 10.1038/nmeth.2524 · Source: PubMed
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Directed differentiation protocols enable derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) and permit engineering of human myocardium in vitro. However, hPSC-derived cardiomyocytes are reflective of very early human development, limiting their utility in the generation of in vitro models of mature myocardium. Here we describe a platform that combines three-dimensional cell cultivation with electrical stimulation to mature hPSC-derived cardiac tissues. We used quantitative structural, molecular and electrophysiological analyses to explain the responses of immature human myocardium to electrical stimulation and pacing. We demonstrated that the engineered platform allows for the generation of three-dimensional, aligned cardiac tissues (biowires) with frequent striations. Biowires submitted to electrical stimulation had markedly increased myofibril ultrastructural organization, elevated conduction velocity and improved both electrophysiological and Ca(2+) handling properties compared to nonstimulated controls. These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation rate.
© 2013 Nature America, Inc. All rights reserved.
Directed differentiation protocols enable derivation of
cardiomyocytes from human pluripotent stem cells (hPSCs) and
permit engineering of human myocardium in vitro. However,
hPSC-derived cardiomyocytes are reflective of very early
human development, limiting their utility in the generation
of in vitro models of mature myocardium. Here we describe
a platform that combines three-dimensional cell cultivation
with electrical stimulation to mature hPSC-derived cardiac
tissues. We used quantitative structural, molecular and
electrophysiological analyses to explain the responses of
immature human myocardium to electrical stimulation and
pacing. We demonstrated that the engineered platform allows
for the generation of three-dimensional, aligned cardiac
tissues (biowires) with frequent striations. Biowires submitted
to electrical stimulation had markedly increased myofibril
ultrastructural organization, elevated conduction velocity
and improved both electrophysiological and Ca
properties compared to nonstimulated controls. These changes
were in agreement with cardiomyocyte maturation and were
dependent on the stimulation rate.
As adult human cardiomyocytes are essentially postmitotic, the
ability to differentiate cardiomyocytes from human embryonic
stem cells (hESCs) and human induced pluripotent stem cells
represents an exceptional opportunity to create
in vitro models of healthy and diseased human cardiac tissues that
can also be patient-specific
and useful in screening new thera-
peutic agents for efficacy. However, differentiated cells exhibit
a low degree of maturation
and are appreciably different from
adult cardiomyocytes.
hESC-derived cardiomyocytes exhibit immature sarcomere
structure characterized by the absence of H zones, I bands
and M lines (day 40 embryoid bodies
), high proliferation rates
Biowire: a platform for maturation of human
pluripotent stem cell–derived cardiomyocytes
Sara S Nunes
, Jason W Miklas
, Jie Liu
, Roozbeh Aschar-Sobbi
, Yun Xiao
, Boyang Zhang
Jiahua Jiang
, Stéphane Massé
, Mark Gagliardi
, Anne Hsieh
, Nimalan Thavandiran
, Michael A Laflamme
Kumaraswamy Nanthakumar
, Gil J Gross
, Peter H Backx
, Gordon Keller
& Milica Radisic
(~17% proliferating cells for day 37 embryoid bodies
and ~10% for day 21–35 embryoid bodies
), immature action
and Ca
handling properties
with contraction
shown to be, in many cases, dependent on trans-sarcolemmal
influx and not on sarcoplasmic reticulum Ca
hESC-based engineered cardiac tissues also exhibit characteris-
tics of immature cells, including immature sarcomere structure
high proliferation rates (15–45% proliferating cells in ref. 14 and
10–30% proliferating cells in ref. 15) and expression of the fetal
gene program
. This is an important caveat when using these
cells as models of adult human tissue
During embryonic development, cardiac cells are exposed to
environmental cues such as extracellular matrix, soluble factors,
mechanical signals and electrical fields that may determine the
emergence of spatial patterns and aid in tissue morphogenesis
Exogenously applied electrical stimulation has also been shown
to influence cell behavior
We created a platform that combines architectural and electrical
cues to generate a microenvironment conducive to maturation of
three-dimensional (3D) hESC-derived and hiPSC-derived cardiac
tissues, termed ‘biowires’. We seeded cells in collagen gel around
a template suture in a microfabricated well and subjected them
to electrical field stimulation with a progressive increase in fre-
quency. Consistent with maturation, stimulated biowires exhib-
ited cardiomyocytes with a remarkable degree of ultrastructural
organization, improved conduction velocity and enhanced Ca
handling and electrophysiological properties.
Engineering of human cardiac biowires
We generated 3D, self-assembled cardiac biowires by seeding
cardiomyocytes, derived from hPSCs using a directed differ-
entiation protocol in embryoid bodies
, and supporting cells
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
Toronto General Research Institute, University Health
Network, Toronto, Ontario, Canada.
Department of Physiology and Medicine, University of Toronto, Toronto, Ontario, Canada.
Cardiology Division, Hospital for
Sick Children, Toronto, Ontario, Canada.
The Toby Hull Cardiac Fibrillation Management Laboratory, Toronto General Hospital, Toronto, Ontario, Canada.
Centre for Regenerative Medicine, University Health Network, Toronto, Ontario, Canada.
Department of Pathology, University of Washington, Seattle, Washington,
Physiology and Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada.
Department of Pediatrics, University of
Toronto, Toronto, Ontario, Canada.
The Heart and Stroke/Richard Lewar Centre of Excellence, Toronto, Ontario, Canada.
Division of Cardiology, University Health
Network, Toronto, Ontario, Canada.
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada.
These authors
contributed equally to this work. Correspondence should be addressed to M.R. (
Received 6 FebRuaRy; accepted 17 May; published online 23 june 2013; doi:10.1038/nMeth.2524
© 2013 Nature America, Inc. All rights reserved.
(fibroblasts, endothelial cells and smooth muscle cells) into a
template poly(dimethylsiloxane) (PDMS) channel, around a
sterile surgical suture in type I collagen gels (Fig. 1 and
Supplementary Fig. 1a). The biowire suture remained anchored
to the device platform during matrix remodeling. Seeded cells
remodeled and contracted the collagen gel matrix during the
first week after seeding (Fig. 1a and Supplementary Fig. 1a)
with ~40% gel compaction (Fig. 1b; final width, ~600 µm).
This allowed us to remove the biowire from the PDMS template
(Supplementary Figs. 1 and 2).
Histological analysis revealed cell alignment along the axis of
the suture (Fig. 1c and Supplementary Fig. 2b). Biowires beat
synchronously and spontaneously between 2 d and 3 d after
seeding and kept beating after gel compaction, demonstrat-
ing that the setup enabled electromechanical cell coupling
(Supplementary Video 1). Biowires could be electrically paced
and responded to physiological agonists such as epinephrine
(β-adrenergic stimulation) by increasing frequency of spontane-
ous beating (Fig. 1d).
After preculture for 1 week, we either submitted the biowires to
electrical field stimulation or cultured them without stimulation
(nonstimulated controls) for 7 d. To assess whether effects were
dependent on stimulation rate, we used two different protocols:
(i) low-frequency ramp-up regimen, where stimulation started
at 1 Hz, increased to 3 Hz (1 Hz, 1.83 Hz, 2.66 Hz and 3 Hz on
days 1–4, respectively) and maintained at 3 Hz for the remainder
of the week (Supplementary Fig. 1b; referred to as low-frequency
regimen or 3-Hz regimen) or (ii) high-frequency ramp-up regi-
men, where stimulation started at 1 Hz and increased to 6 Hz
throughout the week (1 Hz, 1.83 Hz, 2.66 Hz, 3.49 Hz, 4.82 Hz,
5.15 Hz and 6 Hz; Supplementary Fig. 1c; referred to as high-
frequency regimen or 6-Hz regimen).
Physiological hypertrophy in stimulated biowires
After 2 weeks in culture, cells throughout the biowires strongly
expressed cardiac contractile proteins sarcomeric α-actinin,
actin and cardiac troponin T, as evidenced by immunostaining
(Fig. 2a and Supplementary Figs. 2 and 3). Sarcomeric banding
Day 0
200 µm
Day 1 Day 2 Day 4 Day 5 Day 7
Spontaneous activity
1 mm
Electrical stimulation
1 s
+ Epinephrine
b c
Width (µm)
0 1 2
Time in culture (d)
5 6 7
100 µm
Figure 1
Generation of human cardiac biowires. (a) Brightfield images
of Hes2 hESC-derived cardiomyocytes on indicated days of preculture in
biowire template. (b) Quantification of gel compaction on the indicated
days of culture (average ± s.d., n = 3 (day 0), n = 4 (days 1–7)).
(c) Hematoxylin and eosin (H&E) and Masson’s trichrome (MT) staining
of biowire sections (double-headed arrows represent suture axis).
(d) Representative picture (left) of a biowire being imaged with
potentiometric fluorophore (DI-4-ANEPPS), which shows spontaneous
electrical activity, with impulse propagation recording (left trace
recording), response to electrical stimulation (middle trace recording,
stimulation frequency is depicted in red trace below) and increase in
frequency of spontaneous response under pharmacological stimulation
(epinephrine, right trace recording).
Sarcomere Desmosomes
Control3 Hz6 Hz
I bands (per Z disc)
Control 3 Hz 6 Hz
H zones (per sarcomere)
P = 0.01
P = 0.003
Control 3 Hz 6 Hz
P = 0.005
Desmosomes (number
per nm membrane × 10
3 Hz
6 Hz
P = 0.0003
α-Actinin, actin
α-actinin, DAPI
Cardiac troponin T
a b
EBd34 Control
3 Hz 6 Hz
3 Hz6 Hz
Cell shape (%)
Control 3 Hz 6 Hz EBd34
P = 0.01
P = 0.03
Figure 2
Cultured biowires in combination with electrical stimulation
promoted physiological cell hypertrophy and improved cardiomyocyte
phenotype. (a) Representative confocal images of nonstimulated (control)
and electrically stimulated biowires (3-Hz and 6-Hz regimens) showing
cardiomyocyte alignment and frequent Z disks (double-headed arrows
represent suture axis). (b) Analysis of cardiomyocyte cell shape in
indicated conditions (average ± s.d., n = 3 per group). EBd34 versus cells
subjected to 3-Hz regimen, P = 0.01 for both rod-like and round; EBd34
versus 6 Hz, P = 0.03 for both round and rod-like. DAPI, 4,6-diamidino-2-
phenylindole. (c) Representative ultrastructural images of nonstimulated
(control) and electrically stimulated biowires showing sarcomere structure
(sarcomere, white bar; Z disks, black arrows; H zones, white arrows;
m, mitochondria) and presence of desmosomes (desmosomes, white
arrows). Scale bars, 20 µm (a), 50 µm (b) and 1 µm (c). (d) Morphometric
analysis (average ± s.d.; n = 4 per group) showing ratio of H zones to
sarcomeres (control versus 6 Hz, P = 0.005) ratio of I bands to Z disks
(control versus 3 Hz, P = 0.01; control versus 6 Hz, P = 0.003) and number
of desmosomes per membrane length (control versus 6 Hz, P = 0.0003).
*, significant difference between group and control. In normal adult cells,
the ratio of H zones to sarcomeres is 1 and of I bands to Z disks is 2.
Data in ad were obtained with Hes2 hESC-derived cardiomyocytes.
© 2013 Nature America, Inc. All rights reserved.
of the contractile apparatus (Fig. 2a and Supplementary Figs. 2c,
3a and 4) and myofibrillar alignment along the suture axis was
qualitatively similar to the structure in the adult heart
. Biowires
kept in culture for 3 weeks and 4 weeks maintained cell alignment
and their contractile apparatus structure, as evidenced by confocal
and transmission electron microscopy (Supplementary Fig. 5).
Early in cardiac development, cardiomyocytes are round cells
and differentiate into rod-like cells after birth
. Adult human
cardiomyocytes have a structurally rigid architecture, retain-
ing a rod-like shape
immediately after dissociation, whereas
hESC-derived cardiomyocytes remain round. We dissociated
age-matched, EBd34 and biowires, and seeded the cells into
Matrigel-coated plates (Supplementary Fig. 1d). Although ~80%
of cardiomyocytes from EBd34 exhibited a round phenotype, this
percentage was significantly lower (~50% less) in electrically
stimulated samples (Fig. 2b). The percentage of rod-like car-
diomyocytes was significantly higher (about fourfold) in electri-
cally stimulated biowires (Fig. 2b and Supplementary Fig. 6) as
compared to EBd34.
During development, cardiomyocytes undergo physiological
hypertrophy characterized by an increase in cell size followed
by changes in sarcomere structure and downregulation of fetal
. We observed a significant increase in cardiomyocyte
size (area of plated cells) in biowire conditions compared to
cardiomyocytes from age-matched embryoid bodies (EBd34)
(Supplementary Table 1; EBd34 versus control, P = 0.034; EBd34
versus 3-Hz regimen, P = 0.003; EBd34 versus 6-Hz regimen,
P = 0.01). Atrial natriuretic peptide (NPPA), brain natriuretic
peptide (NPPB) and α-myosin heavy chain (MYH6) are proteins
highly expressed in fetal cardiomyocytes and upregulated during
pathological hypertrophy in diseased adult ventricular cardiomyo-
cytes. Downregulation of the fetal cardiac gene program (NPPA,
NPPB and MYH6) in hESC-derived cardiomyocyte biowires
(Supplementary Fig. 7), compared to age-matched embryoid
bodies, in concert with cell-size increase, suggested physiologi-
cal hypertrophy and a more mature phenotype. Although we
observed downregulation of mRNA encoding structural pro-
teins in biowires compared to embryoid bodies, we observed
no changes in the amounts of these protein (Supplementary
Results). Potassium inwardly rectifying channel gene (KCNJ2),
that has important roles in cell excitability and K
, was upregulated in cells from biowires compared to EBd34
(Supplementary Fig. 7).
hESC-derived cardiomyocytes cultured in biowires also
exhibted lower proliferation rates than those of embryoid bodies
(Supplementary Fig. 8; EBd20 versus EBd34, P = 0.002; EBd34
versus control, P = 0.019; EBd34 versus 3-Hz regimen, P = 0.016;
EBd34 versus 6-Hz regimen, P = 0.015) and the percentage of
cardiomyocytes in each condition remained unchanged after cul-
ture for 2 weeks (48.2% ± 10.7% average ± s.d.; Supplementary
Fig. 9). After cultivation, cell composition in biowires was compa-
rable to that in EBd20, specifically CD31
(2.4% ± 1.5%, endothe-
lial cells
), CD90
(34.4% ± 23%, fibroblasts
), calponin
(35 ±
22%, smooth muscle cells) or vimentin
(80% ± 22%, nonmyo-
cytes) cells. This suggests that the observed improvements were
not related to the induction of a particular cell type in biowires.
Maturation of contractile apparatus
Cells in nonstimulated biowires exhibited well-defined Z discs
and myofibrils (Fig. 2c and Supplementary Figs. 3c and 4) but
no signs of alignment of Z discs. In contrast, biowires stimu-
lated under the high-frequency regimen showed signs of mat-
uration, such as organized sarcomeric banding with frequent
myofibrils that converged and displayed aligned Z discs (6-Hz
regimen; Fig. 2c and Supplementary Figs. 3c and 4), many mito-
chondria (6-Hz regimen; Fig. 2c and Supplementary Fig. 3c
and 4) and desmosomes, a molecular complex of cell-cell adhesion
proteins (Fig. 2c). In the 6-Hz regimen condition, mitochondria
were positioned closer to the contractile apparatus than in con-
trol or 3-Hz regimen conditions (Fig. 2c and Supplementary
Figs. 3c and 4b).
Electrically stimulated samples exhibited a sarcomeric organiza-
tion more compatible with mature cells than with nonstimulated
controls as shown by a significantly higher presence of H zones
per sarcomere (Fig. 2d, control versus 6-Hz regimen, P = 0.005;
Supplementary Fig. 3d, control versus 6-Hz regimen, P = 0.001)
and I bands per Z disc (Fig. 2d, control versus 3-Hz regimen,
P = 0.01; control versus 6-Hz regimen, P = 0.003; Supplementary
Fig. 3d, control versus 6-Hz regimen, P = 0.0004). Biowires stim-
ulated using the 6-Hz regimen also had a significantly higher
number of desmosomes per membrane length than did both
nonstimulated controls and biowires subjected to the 3-Hz regi-
men (Fig. 2d, P = 0.0003). In hiPSC-derived cardiomyocyte bio-
wires, we frequently saw areas with nascent intercalated discs
(Supplementary Fig. 3c and 4b). However, the lack of M lines
and T tubules, consistent with previous reports
, indicated an
absence of terminal differentiation.
Functional assessment of engineered biowires
Electrical stimulation using the 6-Hz regimen improved biowires
electrical properties, leading to a significant decrease in the exci-
tation threshold (Fig. 3a; control versus 6-Hz regimen, P = 0.03)
and an increase in the maximum capture rate (MCR) (Fig. 3b,
Control 3 Hz 6 Hz
Local activation
time (ms)
1 mm
Excitation threshold
(V cm
3 Hz
6 Hz
P = 0.03
Maximum capture rate
P = 0.022
3 Hz
6 Hz
Conduction velocity
(cm s
P = 0.014
P = 0.011
3 Hz
6 Hz
Figure 3
Functional assessment of engineered biowires. (ac) Excitation
threshold (a; control (n = 4) versus cells subjected to 6-Hz regimen (n = 3),
P = 0.03; measured by field stimulation and videomicroscopy; 3 Hz,
n = 3), maximum capture rate (b; control versus 6 Hz (n = 4 per group),
P = 0.022; measured by point stimulation and optical mapping; 3 Hz,
n = 3) and electrical impulse propagation rates (c; control (n = 13) versus
3 Hz (n = 10), P = 0.014; control versus 6 Hz (n = 5), P = 0.011; measured
by point stimulation and optical mapping) after electrical stimulation
(average ± s.d.). (d) Representative conduction velocity activation maps
in biowires. *, significant difference between group and control. Data in
ad were obtained with hESC-derived cardiomyocytes from Hes2 cell line.
© 2013 Nature America, Inc. All rights reserved.
control versus 6-Hz regimen, P = 0.022; Supplementary Figs. 3
and 4) as analyzed by point stimulation at the end of the cultiva-
tion in conjunction with optical mapping of impulse propaga-
tion (Supplementary Fig. 10a and Supplementary Videos 25).
Optical mapping demonstrated higher MCR with field stimula-
tion (5.2 Hz) than with point stimulation (4 Hz) (Supplementary
Fig. 10b; we observed an MCR of 5.2 Hz with field stimulation
and intermittent capture at 6 Hz, Supplementary Videos 69).
During field stimulation, all cells received the stimulus at the same
time, and response was not limited by each cell’s propagation limi-
tations. Conduction velocity, assessed upon point stimulation at
the end of cultivation, was ~40% and ~50% higher in the samples
electrically stimulated during culture (3 Hz and 6 Hz, respec-
tively), than nonstimulated controls (Fig. 3c,d, control versus
3 Hz, P = 0.014; control versus 6 Hz, P = 0.011). Improvements
in electrical properties (excitation threshold, MCR and conduc-
tion velocity) were more pronounced with the high-frequency
regimen compared to the low-frequency one. Improvement in
conduction velocity directly correlated with the average number
of desmosomes (Supplementary Fig. 11, R
= 0.8526).
Stimulation improves Ca
-handling properties
Either all
or the majority
of hESC-derived cardiomyocytes rely
on Ca
influx from sarcolemma rather than on Ca
release from
sarcoplasmic reticulum for contraction, which differs markedly
from the case in the adult myocardium. We tested the effect of caf-
feine, which opens sarcoplasmic reticulum ryanodine channels,
on cytosolic Ca
transients in single cells isolated from biowires
(Supplementary Fig. 1d). In accordance with previous work
none of the hESC-derived cardiomyocytes in nonstimulated
controls were responsive to caffeine (Fig. 4a), whereas electri-
cally stimulated cells using both 3-Hz and 6-Hz regimen condi-
tions responded to caffeine by inducing an increase in cytosolic
(Fig. 4b,c). Quantification of Ca
transient amplitudes
showed that electrically stimulated cells exhibited significantly
higher amplitude intensity in response to caffeine than nonstim-
ulated controls, in a stimulation frequency–dependent manner
(Fig. 4d,e). Blockade of L-type Ca
channels in cells from bio-
wires subjected to the 6-Hz regimen with either verapamil or
nifedipine (Fig. 4f,g) led, as expected in mature cells, to cessa-
tion of Ca
transients. Addition of caffeine after blockade of
L-type Ca
channels led to Ca
release into the cytosol (Fig. 4f,g).
Blockade of the ion-transport activity of sarcoplasmic reticulum
ATPase (SERCA) by addition of thapsigargin (Fig. 4h) led to
the cessation of calcium transients with time because of the deple-
tion of Ca
from the sarcoplasmic reticulum. Cardiomyocytes
from the 6-Hz regimen condition also demonstrated a faster ris-
ing slope and time to peak, parameters that represent the kinetics
of Ca
release into the cytosol and faster τ decay and time to base,
parameters that represent the kinetics of clearance of Ca
the cytosol (Supplementary Table 2). Taken together, these data
indicated that cardiac biowires stimulated using the 6-Hz regi-
men during culture exhibited Ca
-handling properties compat-
ible with a functional sarcoplasmic reticulum.
Stimulation alters electrophysiological properties
To assess maturity, we measured action potentials, human ERG
(hERG) currents and inward rectifier currents (I
in cardio-
myocytes derived from biowires and embryoid bodies (Fig. 5).
hERG currents were larger (P = 0.0434) in biowires subjected to
the 6-Hz regimen (0.81 pA pF
± 0.09 pA pF
) than nonstimu-
lated controls (0.52 pA pF
± 0.10 pA pF
) (Fig. 5a) without dif-
ferences in their biophysical properties (Supplementary Fig. 12).
Cardiomyocytes from both biowire groups had higher hERG levels
compared to those from day-20 or day-44 embryoid bodies (Fig. 5a).
Similarly, I
densities were greater (P = 0.0406) in biowires
subjected to the 6-Hz regimen (1.53 pA pF
± 0.25 pA pF
than in controls (0.94 pA pF
± 0.14 pA pF
), and I
in both biowire groups were greater (P = 0.0005) than those
recorded in embryoid body–derived cardiomyocytes (Fig. 5b).
Caffeine, 5 mM
Verapamil, 1 mM
20 s
1.0 F/F0
]i transients
Caffeine, 5 mM
Nifedipine, 10 µM
20 s
1.0 F/F0
]i transients
Caffeine, 5 mM
Thapsigargin, 2 µM
20 s
1.0 F/F0
]i transien
Caffeine, 5 mM
Caffeine, 5 mM
20 s
1.0 F/F0
]i transients
Caffeine, 5 mM
20 s
1.0 F/F0
]i transients
Caffeine, 5 mM
20 s
1.0 F/F0
]i transients
3 Hz
6 Hz
Caffeine-induced change
of peak intensity (F, %)
Figure 4
Electrical stimulation promoted
improvement in Ca
handling properties.
(ac) Representative traces of Ca
in response to caffeine in nonstimulated
control cells (a), cells subjected to the
3-Hz regimen (b) and 6-Hz regimen (c).
(d) Caffeine-induced change of peak
fluorescence intensity among experimental
groups (mean ± s.e.m. after normalizing the
peak fluorescence intensity before administration
of caffeine; control versus 3 Hz, P = 1.1 × 10
; control versus 6 Hz, P = 2.1 × 10
; 3 Hz versus 6 Hz, P = 0.003; n = 10 (control), n = 6 (3 Hz) and
n = 9 (6 Hz)). (e) Representative fluorescence recording of Ca
transients before and after administration of caffeine at 5 mM (arrow) in cells subjected
to the 6-Hz regimen. (fh) Inhibition of L-type Ca
channels with verapamil (f) or nifedipine (g) and blockade of SERCA channels with thapsigargin (h)
in cells subjected to the 6-Hz regimen before addition of caffeine. *, significant difference between group and control.
, significant difference between
3-Hz regimen group and 6-Hz regimen group. Data in ah were obtained with hESC-derived cardiomyocytes obtained from Hes2 cell line and represent
measurements performed in single cell cardiomyocytes after dissociation from biowires. F, fluorescence intensity; F0, fluorescence intensity at baseline;
F/F0, F normalized by F0.
© 2013 Nature America, Inc. All rights reserved.
Cell capacitance, a measure of cell size, was larger (P = 0.0052)
in biowires subjected to the 6-Hz regimen (19.59 pF ± 1.41 pF)
compared to control biowires (14.23 pF ± 0.90 pF) and smaller
(P = 0.0041) in embryoid body–derived cardiomyocytes (Fig. 5c).
Resting membrane potentials (V
) of the cardiomyocytes from
biowires were more negative than in embryoid body–derived car-
diomyocytes (P < 0.0001; Fig. 5d). After correcting for the liquid
junction potential, which was ~16 mV, the values of V
in biowire cardiomyocytes with the patch-clamp method were
well below the equilibrium potential for Nernst potential for K
= −96 mV), suggesting that hyperpolarizing currents, possibly
those generated by the Na
, strongly influenced V
Consistently, we found that the cardiomyocytes from biowires
had a very low resting membrane conductance, which correlated
with V
(R = 0.5584, P < 0.0001), and I
values had negative
correlations with V
(R = 0.2267, P = 0.0216; Supplementary
Fig. 13). Maximum depolarization rates (Fig. 5e) and peak volt-
ages of the action potentials (Fig. 5f) did not differ between the
two biowire groups. However, both properties were improved in
these biowire groups compared to embryoid bodies (P = 0.5248
and P = 0.0488, respectively). Action potential durations were
longer (P = 0.0021) with greater variation in embryoid body–
derived cardiomyocytes than biowire-derived cardiomyocytes
(Fig. 5g and Supplementary Fig. 14), suggesting less electrophys-
iological diversity and more maturation in biowires. Automaticity
(spontaneous beating activity) was greater (P = 0.0414) in embry-
oid body–derived cardiomyocytes compared to control biowires
(Fig. 5h), which was comparable to that in biowires subjected
to the 6-Hz regimen. Taken together, these results support the
conclusion that biowires and electrical stimulation using the 6-Hz
regimen promoted electrophysiological maturation.
Normal human fetal heart rate varies but is maintained at ~3 Hz
for most of the time
whereas adult resting heart rate is ~1 Hz
The rate change is associated with changes in expression of con-
tractile proteins and suggests a possible dependence of cardiac
maturation on stimulation rate. The fact that the progressive
increase from 1 Hz to 6 Hz was the best stimulation condition
tested in biowires was a surprise to us as 3 Hz is the average fetal
heart rate
. This could be a compensatory mechanism for the
lack of other important cells types and cell-cell developmental
guidance in the in vitro setting. As we increased frequency of field
stimulation over 7 d in culture, the group subjected to the 6-Hz
regimen might only lose capture (exceed the rate of 5.2 Hz) at
the very last day of stimulation. Therefore, it may be the stimula-
tion at the highest possible rate, and not the rate itself, that is the
governing cue for cardiomyocyte maturation in vitro.
Mechanical stimulation has been reported to lead to a robust
induction of structural proteins such as myosin heavy chain and
induce proliferation of hPSC-derived cardiomyocytes
, sug-
gesting that electrical stimulation of the biowire at 6 Hz did not
simply provide a better mechanical stimulation environment.
Previously, mechanical stimulation did not lead to electrophysio-
logical maturation
. The use of electrical stimulation in conjunc-
tion with stretch as a mimic of cardiac load
, concurrently or
sequentially, might be required to induce terminal differentiation
in hPSC-derived cardiomyocytes and upregulate the expression of
myofilament proteins. Other strategies might include cultivation
in the presence of T3 thyroid hormone
, insulin-like growth
factor-1 (ref. 36), addition of laminin or native decellularized
heart extracellular matrix into the hydrogel mixture
cultivation on stiffer substrates
It is well accepted that some human stem cell lines are more
cardiomyogenic than others
, and these differences could
also be related to the maturity of the produced cells. In previous
, many and usually most cells were irresponsive to
caffeine at the end of differentiation. Therefore, differences in Ca
handling properties could also be due to cell line variability. Here we
demonstrated that in a given cell line, culture in biowires and electri-
cal-field stimulation enhanced Ca
-handling properties of cardio-
myocytes consistent with a functional sarcoplasmic reticulum.
Biowire cardiomyocytes were clearly more mature than
cardiomyocytes obtained from embryoid bodies cultivated for
20 d (EBd20) or for 40–44 d (EBd44), which exhibited a greater
propensity for automaticity, more depolarized membrane
Action potential
Peak voltage (mV)
6 Hz Control EBd44 EBd20
n = 17
n = 13 n = 13 NA
P = 0.0200
P = 0.0488
at –100 mV
current density (pA/pF)
6 Hz Control EBd44 EBd20
n = 25 n = 14
n = 28 n = 15
P = 0.0406
P = 0.0471
P = 0.0434
6 Hz
hERG current density
Control EBd44 EBd20
n = 25 n = 27 n = 16
n = 56
P = 0.0387
P = 0.0355
Resting membrane
potential (mV)
6 Hz Control EBd44 EBd20
n = 36 n = 51
n = 16
n = 13
P < 0.0001
P < 0.0001
Cell capacitance (pF)
6 Hz Control EBd44 EBd20
n = 62 n = 60n = 42 n = 16
P = 0.0052
P = 0.0048
APD90 (ms)
6 Hz Control EBd44 EBd20
n = 17 n = 13 n = 13 NA
P = 0.0021
Maximum depolarization
rate (mV ms
6 Hz Control EBd44 EBd20
n = 17
n = 13 n = 13 NA
P = 0.0107
Automaticity No automaticity
Ratio (%)
6 Hz Control EBd44 EBd20
n = 5 n = 5 n = 11
P = 0.0414
n = 12 n = 8 n = 2
Figure 5
Electrophysiological properties in single cardiomyocytes isolated from biowires or embryoid bodies and recorded with patch clamp. (a) hERG
tail current density. (b) I
current density measured at −100 mV. (c) Cell capacitance. (d) Resting membrane potential. (e) Maximum depolarization
rate of action potential. (f) Action potential peak voltage. (g) Action potential duration measured at 90% repolarization (APD90). (h) Ratio of cells
displaying spontaneous beating (automaticity) or no spontaneous beating (no automaticity). Control, control biowire. Data in ah were obtained with
hESC-derived cardiomyocytes obtained from Hes2 cell line (average ± s.e.m.).
© 2013 Nature America, Inc. All rights reserved.
potentials, lower cell capacitance and less hERG currents and I
Electrophysiological measurements of the EBd20 cardiomyocytes
represented the cell properties before their incorporation into
biowires, whereas EBd44 cardiomyocytes were cultured for peri-
ods slightly longer than the biowire culture time, allowing assess-
ment of the independent effect of culture time on maturation
We acknowledge that biowire maturation is clearly incomplete,
as evidenced by the relatively low membrane conductance.
Nevertheless, it is intriguing to speculate that the combination
of low membrane conductance with V
below E
may represent
an ‘intermediate’ phenotype as cardiomyocytes undergo matura-
tion from the embryonic state.
Correlating the properties of hPSC-derived cardiomyocytes in
biowires with mouse or human development could help to gauge
maturation stage, but mouse and rat cardiomyocytes are physi-
ologically distinct, and age-defined healthy human heart samples
are scarce. Additionally, in vitro maturation might not be compat-
ible with embryo development.
The small size (radius of ~300 µm) of biowire upon gel com-
paction was selected to be close to the diffusional limitations
for oxygen supply to ensure that the biowires can be maintained
in culture without perfusion. Addition of vascular cells will be
imperative for improving survival and promoting integration
with the host tissue in future in vivo studies
. We generated a
unique platform that enables generation of human cardiac tissues
of graded levels of maturation that can be used to determine, in
future in vivo studies, the optimal maturation level that will result
in the highest ability of cells to survive and integrate in adult
hearts with the lowest side effects (such as arrhythmias).
Methods and any associated references are available in the online
version of the paper.
Note: Supplementary information is available in the online version of the paper.
We thank P. Lai, C. Laschinger, N. Dubois and B. Calvieri for technical assistance,
C.C. Chang and L. Fu for assistance with biowire setup figure preparation. Funded
by grants from Ontario Research Fund–Global Leadership Round 2 (ORF-GL2),
National Sciences and Engineering Research Council of Canada (NSERC) Strategic
Grant (STPGP 381002-09), Canadian Institutes of Health Research (CIHR)
Operating Grant (MOP-126027 and MOP-62954), NSERC-CIHR Collaborative Health
Research Grant (CHRPJ 385981-10), NSERC Discovery Grant (RGPIN 326982-10),
and NSERC Discovery Accelerator Supplement (RGPAS 396125-10) and National
Institutes of Health grant 2R01 HL076485.
S.S.N. developed biowire concept, designed and performed experiments, analyzed
data and prepared the manuscript. J.W.M. performed experiments and analyzed
data. J.L., R.A.-S. and P.H.B. performed patch clamping and microelectrode
recordings. Y.X. designed and validated initial device. B.Z. designed and fabricated
masters for device fabrication. J.J. and G.J.G. performed calcium transient
measurement and analysis. S.M. and K.N. performed optical mapping measurements
and analysis. M.G. and G.K. differentiated hESC-derived cardiomyocytes.
A.H. designed primers. N.T. developed initial collagen gel mixture. M.A.L. provided
training on hiPSC differentiation and cells. P.H.B. contributed to writing of the
manuscript. M.R. envisioned the biowire concept and electrical stimulation
protocol, supervised the work and wrote the manuscript.
The authors declare competing financial interests: details are available in the
online version of the paper.
Reprints and permissions information is available online at http://www.nature.
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Human pluripotent stem cell culture and differentiation.
Cardiomyocytes derived from two different hESC lines (Hes2 and
Hes3) and two different hiPSC lines (CDI-MRB and HR-I-2Cr-2R)
were used. Both hESC lines and hiPSC line HR-I-2Cr-2R were
maintained as described
. Embryoid bodies (EBs) were differen-
tiated to the cardiovascular lineage as previously described
. In
brief, EBs were generated by culture in StemPro-34 (Invitrogen)
medium containing BMP4 (1 ng/ml). On day 1, EBs were col-
lected and suspended in induction medium (StemPro-34 contain-
ing basic fibroblast growth factor (bFGF; 2.5 ng/ml), activin A
(6 ng/ml) and BMP4 (10 ng/ml)). On day 4, the EBs were collected
from the induction medium and recultured in StemPro-34
supplemented with vascular endothelial growth factor (VEGF;
10 ng/ml) and DKK1 (150 ng/ml). On day 8, the medium was
changed again and the EBs were cultured in StemPro-34 contain-
ing VEGF (20 ng/ml) and bFGF (10 ng/ml) for the duration of
the experiment. Cultures were maintained in hypoxic environ-
ment (5% CO
and 5% O
) for the first 12 d and then transferred
into 5% CO
for the remainder of the culture period. EBs were
dissociated for seeding in biowires on day 20 (EBd20), day 34
(EBd34) and day 40–44 (EBd44) for specific cellular and electro-
physiological analyses. CDI-MRB hiPSC-derived cardiomyocytes
were purchased from Cellular Dynamics International (CMC-
100-110-001) and used for biowire production immediately
after thawing.
Device design and manufacture. The device was fabricated
using soft lithography technique. A two-layer SU-8 (Microchem
Corp.) master was used to mold PDMS. Briefly, device features
were printed on two film masks (CADART) corresponding to the
two-layer design. SU-8 2050 was spun onto 4-inch silicon wafer,
baked and exposed to UV light under the first-layer mask to cre-
ate the first layer including the suture channel and the chamber
with thickness of 185 µm. The second layer including only the
chamber with thickness of 115 µm was spun on top. After addi-
tional baking, the second-layer mask was aligned to the features
on the first layer and then exposed to UV light. Finally, the wafer
was developed using propylene glycol monomethyl ether acetate
(Doe & Ingalls Inc.). PDMS was then cast onto the SU-8 master
and baked for 2 h at 70 °C. PDMS templates were used to hold a
piece of surgical suture centrally in the channel (Supplementary
Fig. 1a) to which the cardiac cell suspension gel was added.
Biowire generation. EBd20 generated as described above were
incubated in collagenase type I (1 mg/ml; Sigma) and DNAse
(1 mg/ml, CalBiochem) in Hanks balanced salt solution (NaCl,
136 mM; NaHCO
, 4.16 mM; Na
, 0.34 mM; KCl, 5.36 mM;
, 0.44 mM; dextrose, 5.55 mM and HEPES, 5 mM) for
2 h at 37 °C. EBs were centrifuged (107g, 5 min), incubated with
trypsin (0.25%, Gibco) for 5 min at 37 °C and pipetted gently
to dissociate the cells. After dissociation, cells were centrifuged
(167 x g, 5 min), counted and seeded at 0.5 × 10
cells/wire of
0.5 cm in length. This ratio was maintained for generation of longer
biowires. Cells were seeded in collagen type I gels (4 µl/0.5 cm wire
length; 2.1 mg/ml of rat tail collagen type I (BD Biosciences) in
24.9 mM glucose, 23.81 mM NaHCO
, 14.34 mM NaOH, 10 mM
HEPES, in 1× M199 medium plus 10% of growth factor reduced
Matrigel (BD Biosciences)) by pipetting the cell suspension into
the main channel of the PDMS template (Supplementary Fig. 1a).
CDI-MRB hiPSC-derived cardiomyocytes were thawed,
counted and seeded in same concentration as hESC-derived
cardiomyocytes. After seeding, cells were kept in culture for 7 d
to allow collagen matrix remodeling and assembly around the
suture (Fig. 1a,b).
Electrical stimulation setup and conditions. After preculture for
7 d, biowires were transferred to stimulation chambers fitted with
two 1/4-inch-diameter carbon rods (Ladd Research Industries)
placed 2 cm apart and connected to a cardiac stimulator (Grass
s88x) with platinum wires (Ladd Research Industries). Biowires
were placed perpendicular to the electrodes and were either
submitted to electrical stimulation (rectangular, biphasic, 1 ms,
3–4 V/cm) or cultured without electrical stimulation (nonstimu-
lated controls or control) for 7 d (Supplementary Fig. 1b,c). As
increased time in culture affects maturation
, age-matched EBs
(EBd34) were used as an additional control. For long-term stimu-
lation experiments, shown in Supplementary Fig. 5, the biowires
were precultured for 7 d as described above, followed by 7 d of
6-Hz protocol, at which point the frequency was decreased to
1 Hz (to mimic postnatal decrease in heart rate) and maintained
for an additional 14 d.
To verify that biowire-stimulated cardiomyocytes truly exhib-
ited maturation on a single-cell basis, assays were performed
in which single cells were used. With this goal, biowires were
digested with collagenase type I (1 mg/ml; Sigma) and DNAse
(1 mg/ml, CalBiochem) in Hanks balanced salt solution (NaCl,
136 mM; NaHCO
, 4.16 mM; Na
, 0.34 mM; KCl, 5.36 mM;
, 0.44 mM; dextrose, 5.55 mM; and HEPES, 5 mM) for
4 h at 37 °C, centrifuged (107g, 5 min), incubated with trypsin
(0.25%, Gibco) for 5 min at 37 °C and pipetted gently to dissoci-
ate the cells as depicted in Supplementary Figure 1d. Isolated
single cells were seeded on Matrigel-coated or laminin-coated
glass coverslips as described below, and area, calcium transient
and patch-clamp measurements were performed.
Assessments. The progression of tissue assembly was assessed
at various levels after 2 weeks in culture (7 d of gel compaction
followed by 7 d of stimulation): functional (excitation threshold,
MCR, conduction velocity and Ca
handling); ultrastructural
(sarcomere development, frequency (number/membrane length)
of desmosomes), cellular (cell size and shape, proliferation, dis-
tribution of cardiac proteins: actin, troponin T and α-actinin),
electrophysiological (hERG, I
and I
) and molecular (expres-
sion of cardiac genes and proteins).
Immunostaining and fluorescence microscopy. Immunostaining
was performed using the following antibodies: mouse anti
cardiac troponin T (1:100, Thermo Scientific; MS-295-P1), mouse
anti-α-actinin (1:200, Abcam, ab9465), anti-mouse–Alexa Fluor
488 (1:400, Invitrogen, A21202), anti-Ki67 (1:250, Millipore,
AB9260), anti-rabbit–TRITC (1:400, Invitrogen, 81-6114).
DAPI was used to counterstain nuclei. Phalloidin–Alexa Fluor
660 (1:1,000, Invitrogen, A22285) was used to detect actin fibers.
The stained cells were visualized using a fluorescence microscope
(Leica CTR6000) and images captured using the Leica Application
Suite software. For confocal microscopy, cells were visualized
using a fluorescence confocal microscope (Zeiss LSM-510).
© 2013 Nature America, Inc. All rights reserved.
Transmission electron microscopy. Tissue was fixed with
4% paraformaldehyde, 1% glutaraldehyde in 0.1 M PBS for at
least 1 h and washed 3 times with PBS pH 7.2. Post-fixation was
done with 1% osmium tetraoxide in 0.1 M PBS, pH 7.2 for 1 h
and dehydrated using ethanol series from 25% to 100%. Tissue
was infiltrated using Epon resin and polymerized in plastic dishes
at 40 °C for 48 h. Tissue was stained with uranyl acetate and lead
citrate after sectioning. Imaging was performed at Hitachi H-7000
transmission electron microscope.
Optical mapping. Biowires were incubated with a voltage-
sensitive dye (Di-4-ANEPPS, 5 µM, Invitrogen) for 20 min at
37 °C in warm Tyrodes solution (118 mM NaCl, 4.7 mM KCl,
1.25 mM CaCl
, 0.6 mM MgSO
, 1.2 mM KH
, 25 mM
and 6 mM glucose; oxygenated by bubbling carbogen
95% O
, 5% CO
for at least 20 min shortly before use). Dye
fluorescence was recorded on an MVX-10 Olympus fluorescence
microscope equipped with a high-speed complementary metal-
oxide semiconductor (CMOS) camera (Ultima-L, Scimedia)
The 1-cm sensor had 100 × 100 pixel resolution and the spa-
tial resolution was 50–100 µm/pixel. Imaging was performed at
200 frames/s. The fluorescence was excited using a mercury arc
source (X-Cite Exacte) with green filter (Olympus U-MWIG2
filter cube). The constructs were electrically point-stimulated
using a bipolar electrode made of two fine wires (AWG#32)
inserted in a stainless steel needle, which was mounted on a
micromanipulator (World Precision Instruments). For electri-
cal field stimulation, the chamber depicted in Supplementary
Figure 1 was used. The plate containing the biowires was placed
on a heated plate (MATS-U55S, Olympus) and temperature was
regulated at 38 °C. Data analysis was performed using BrainVision
software (Scimedia).
Intracellular recordings. Action potentials were recorded in bio-
wires with high-impedance glass microelectrodes (50–70 M,
filled with 3 M KCl) at 37 ± 0.5 °C. Biowires were superfused
with Krebs solution containing 118 mM NaCl, 4.2 mM KCl,
1.2 mM KH
, 1.8 mM CaCl
, 1.2 mM MgSO
, 23 mM
, 20 mM glucose and 2 mM Na-pyruvate, equilibrated
with 95% O
and 5% CO
; final pH was 7.4. The microelectrodes
were connected to an Axopatch 200B amplifier (Axon Instrument)
current clamp. Signals were filtered at 1 kHz, sampled at 2 kHz and
analyzed with Clampfit 10 (Axon Instrument). Resting potential
was measured at I = 0 mode. For some experiments, biowires were
paced using field stimulation set at twice the excitation threshold.
Patch-clamp recordings. Single cells isolated from biowires
(Supplementary Fig. 1d) or EBs were seeded on laminin-coated
glass coverslips (laminin, Sigma-Aldrich, 10 µg/cm
) overnight
before patch-clamp experiments were performed. Whole-cell
patch-clamp recordings were made using an Axopatch 200B
amplifier at room temperature (23–25 °C). Data were analyzed
with Clampfit 8.0 (Axon Instrument). Amplifier was set at I = 0
when measuring resting potential of cells. Action potentials were
recorded by using the current-clamp mode method. Myocytes
were stimulated at 1 Hz, and the maximum rate of membrane
depolarization, the action potential peak and action potential
duration at 90% (APD90) of the 10th action potential were mea-
sured. The membrane potentials were not corrected for the liquid
junction potentials, which were estimated to be 15.9 mV (esti-
mated with Clampfit 8.0) for the solutions used. Na
hERG current and I
current were also recorded under voltage-
clamp conditions with 70–80% series resistance compensation.
current was induced from holding potential of80 mV by
applying a series of test pulses ranging from −120 mV to +30 mV
for 500 ms with 10-mV increments followed by a test pulse to
−10 mV for 100 ms for measurement of steady-state inactiva-
tion. Although this protocol simultaneously activates overlapping
voltage-dependent Ca
currents, these Ca
currents were esti-
mated (using prepulse protocols) to be less than 3% of the evoked
currents. hERG was assessed by measuring tail currents in
response to steps to −50 mV (for 500 ms) after depolarization to
voltage steps ranging from −45 mV to 60 mV with 15-mV incre-
ments for 2,000 ms. The peak amplitude of hERG tail current
was measured and compared. I
was measured in two ways that
were found to be equivalent for our studies. For complete I-V
relationships, we assessed Ba
-sensitive currents by subtracting
(trace-by-trace for voltage steps ranging from 120 to −10 mV in
10-mV increments from holding potential of −40 mV) the cur-
rents measured in the presence of 500 µM Ba
from the current
measured in the absence of Ba
. For the purposes of measuring
the I
density, we subtracted the background current from that
measured in the absence of Ba
at −100 mV.
Patch-clamp recordings were performed in bath solutions
containing 140 mM NaCl, 4 mM KCl, 1 mM MgCl
, 1.2 mM
, 10 mM HEPES, 10 mM -glucose, at pH 7.35 adjusted
with NaOH. Pipette resistance was ~5.5–7.5 M when filled with
a solution containing 120 mM potassium aspartate, 20 mM KCl,
4 mM NaCl, 1 mM MgCl
, 5 mM MgATP, 10 mM HEPES and
10 mM EGTA, at pH 7.2 adjusted with KOH (calculated reversal
potential of K
was −95.6 mV after pH adjustment). Dofetilide
100 nM (ref. 44) and BaCl
500 µM
were used to block hERG
current and I
, respectively.
Calcium transient measurements. Biowires were dissociated by
incubation with collagenase and trypsin as described above. The
dissociated cardiomyocytes were plated onto 25-mm microscope
glass coverslips coated with growth factor–free Matrigel (diluted
1:60 in RPMI medium) and cultured overnight. Cells were then
incubated with 5 µM of fluo-4 acetoxymethyl ester (fluo-4 AM)
in culture medium for 2 h at 37 °C. Subsequently, cardiomyocytes
were washed twice with dye-free medium and placed back into
the incubator for 30 min. A laser-scanning confocal microscope
(Zeiss LSM 510) was used to measure the fluorescence intensity
of fluo-4 AM. The coverslips containing the fluo-4 AM–loaded
cardiomyocytes were moved onto a special chamber and tightly
secured. Approximately 1.8–1.9 ml of culture medium was added
into the chamber, which was placed on a temperature-controlled
plate (37 °C) on the microscope. Fluo-4 AM was excited via an
argon laser (488 nm), and emitted fluorescence was collected
through a 505 nm emission filter. Changes in fluo-4 AM fluores-
cence intensity, which indicates transient fluctuation of cytosolic
calcium concentration, were recorded in frame and line-scan
model. The images and fluorescence data were acquired through
Zeiss software. The fluorescence data were analyzed with Origin
8.5 software. Fluorescence signals were normalized to baseline
fluorescence after loading fluo-4 AM. The rising phase of the
signals was fitted by linear model, and the decaying phase of the
© 2013 Nature America, Inc. All rights reserved.
signals was fitted by ExpDecay with Offset model. Caffeine, vera-
pamil (Sigma) and fluo-4 AM (Invitrogen) were directly added
into the chamber that contained the cardiomyocytes during imag-
ing at concentrations indicated in the figures. Cells beating at
similar average beating frequency (9.4 ± 0.7 beats per minute
(bpm) for control, 9 ± 0.7 bpm for 3-Hz regimen and 10 ± 0.8 bpm
for 6-Hz regimen) were used for calcium transient measure-
ments to ensure that differences in beating rates would not affect
the measurements.
Quantitative RT-PCR. RT-PCR was performed as previously
. Total RNA was prepared with the High Pure RNA
Isolation Kit (Roche) and treated with RNase-free DNase (Roche).
RNA was reverse-transcribed into cDNA using random hexamers
and oligo(dT) with SuperScript VILO (Invitrogen). RT-qPCR
was performed on a LightCycler 480 (Roche) using LightCycler
480 SYBR Green I Master (Roche). Expression levels were
normalized to the housekeeping genes TATA box binding
protein (TBP) or glyceraldehyde 3-phosphate dehydrogenase
(GAPDH). The oligonucleotide sequences are summarized in
Supplementary Table 3.
Flow cytometry analysis. Cells were obtained from biowires or
EBs by dissociation with collagenase and trypsin as described
above and fixed with 4% paraformaldehyde for 10 min at room
temperature. For intracellular epitopes, cells were permeabilized
in PBS containing 5% FBS and 0.1% Triton X for 10 min on ice
before a blocking step of 5% FBS in PBS for 30 min. Cells were
incubated with the following antibodies in blocking buffer on
ice for 1 h: anti-CD31-PE (1:100) and anti-CD90-APC (1:500,
BD Biosciences, 553373 and 559879, respectively); anti–cardiac
troponin T (1:100, Thermo Scientific, MS-295-P1); anti-calponin
H1 (1:250, Abcam, ab46794); and anti-vimentin (1:100, Sigma-
Aldrich, V6630). Secondary antibodies used were anti-mouse–
Alexa Fluor 488 (1:400, Invitrogen, A21202) and anti-rabbit–Cy5
(1:500, Jackson ImmunoResearch, 111-175-144). Owing to the
intrinsic variability in the percentage of cardiomyocytes in each
assay, the percentage of cells positive for each marker (above the
secondary-antibody-only control) was normalized to the starting
cell population (EBd20) of each experiment to accurately evaluate
whether a change in cell population was occurring.
Western blotting. Biowires were solubilized in (2×) Novex Tris-
Glycine SDS sample buffer (Life technologies) and proteins were
separated by electrophoresis in Novex Tris-Glycine gels (Life
Technologies) and transferred to Biotrace NT (Nitrocellulose,
Pall Corp.). Membranes were probed with either anti-myosin
heavy chain (total, Abcam, ab15, 1:2,000), Phospholamban 1D11
(gift of A. Gramolini, University of Toronto, 1:5,000) or GAPDH
(Millipore, MAB374, 1:10,000) antibodies. Secondary antibod-
ies used were peroxidase-conjugated (DAKO, P0448 or P0447,
1:2000). Membranes were developed with ECL reagent Luminata
Classico Substrate (Millipore).
Statistical analysis. Statistical analysis was performed using
SigmaPlot 12.0. Differences between experimental groups
were analyzed by Student’s t-test except in Figure 2b and
Supplementary Table 1, where statistics was done using two-
way ANOVA. Normality test (Shapiro-Wilk) and pairwise mul-
tiple comparison procedures (Holm-Sidak method) were used
for two-way ANOVA tests. In Supplementary Figure 6, statistics
was done using one-way ANOVA on Ranks (pairwise multiple
comparison, Dunns method). In Figure 5 and Supplementary
Figures 1214, differences between experimental groups were
analyzed by Students t-test, chi-squared test, ANOVA (pairwise
multiple comparisons, Holm-Sidak method). P < 0.05 was con-
sidered significant for all statistical tests.
42. Nanthakumar, K. et al. Optical mapping of Langendorff-perfused human
hearts: establishing a model for the study of ventricular fibrillation in
humans. Am. J. Physiol. Heart Circ. Physiol. 293, H875–H880 (2007).
43. Witkowski, F.X., Clark, R.B., Larsen, T.S., Melnikov, A. & Giles, W.R.
Voltage-sensitive dye recordings of electrophysiological activation in a
Langendorff-perfused mouse heart. Can. J. Cardiol. 13, 1077–1082 (1997).
44. Snyders, D.J. & Chaudhary, A. High affinity open channel block by
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Supplementary resource

  • ... Thus, strategies aiming to differentiate these cells toward a more mature adult-like phenotype would significantly improve their therapeutic value Scuderi and Butcher, 2017;Machiraju and Greenway, 2019;Karbassi et al., 2020). Many approaches have been proposed so far, spanning from the maintenance in culture for an extended period of time (Sartiani et al., 2007;Lundy et al., 2013), to the use of biochemical (i.e., growth factors and co-culture strategies) (Yoshida et al., 2018;Selvaraj et al., 2019) electrical stimuli (Nunes et al., 2013;Hirt et al., 2014) and mechanical load (Leychenko et al., 2011;Haggart et al., 2014); among them, those combining 3D culture and bioengineering methods have been the most effective (Tiburcy et al., 2017;Ronaldson-Bouchard et al., 2018;Ulmer et al., 2018). ...
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    Cardiovascular diseases represent the major cause of morbidity and mortality worldwide. Multiple studies have been conducted so far in order to develop treatments able to prevent the progression of these pathologies. Despite progress made in the last decade, current therapies are still hampered by poor translation into actual clinical applications. The major drawback of such strategies is represented by the limited regenerative capacity of the cardiac tissue. Indeed, after an ischaemic insult, the formation of fibrotic scar takes place, interfering with mechanical and electrical functions of the heart. Hence, the ability of the heart to recover after ischaemic injury depends on several molecular and cellular pathways, and the imbalance between them results into adverse remodeling, culminating in heart failure. In this complex scenario, a new chapter of regenerative medicine has been opened over the past 20 years with the discovery of induced pluripotent stem cells (iPSCs). These cells share the same characteristic of embryonic stem cells (ESCs), but are generated from patient-specific somatic cells, overcoming the ethical limitations related to ESC use and providing an autologous source of human cells. Similarly to ESCs, iPSCs are able to efficiently differentiate into cardiomyocytes (CMs), and thus hold a real regenerative potential for future clinical applications. However, cell-based therapies are subjected to poor grafting and may cause adverse effects in the failing heart. Thus, over the last years, bioengineering technologies focused their attention on the improvement of both survival and functionality of iPSC-derived CMs. The combination of these two fields of study has burst the development of cell-based three-dimensional (3D) structures and organoids which mimic, more realistically, the in vivo cell behavior. Toward the same path, the possibility to directly induce conversion of fibroblasts into CMs has recently emerged as a promising area for in situ cardiac regeneration. In this review we provide an up-to-date overview of the latest advancements in the application of pluripotent stem cells and tissue-engineering for therapeutically relevant cardiac regenerative approaches, aiming to highlight outcomes, limitations and future perspectives for their clinical translation.
  • ... [18,58] Numerous strategies have been developed to improve the maturity of hiPSC-CMs and engineered cardiac tissues. These approaches include varying the culture stiffness, [59] generating biomimetic 3D microenvironment, [60] applying biochemical [61] and electrical stimulation, [62] and combining multiple engineered strategies. [63] Future studies should focus on better understanding of the maturation process, the role of extracellular matrix (ECM) and microenvironment to mimic the physiological environment and promote maturation. ...
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    Ischemic heart disease (IHD) is one of the most common cardiovascular diseases and is the leading cause of death worldwide. Stem cell therapy is a promising strategy to promote cardiac regeneration and myocardial function recovery. Recently, the generation of human induced pluripotent cells (hiPSCs) and their differentiation into cardiomyocytes and vascular cells offer an unprecedented opportunity for the IHD treatment. This review briefly summarizes hiPSCs and their differentiation, and presents the recent advances in hiPSC injection, engineered cardiac patch fabrication, and the application of hiPSC derived extracellular vesicle. Current challenges and further perspectives are also discussed to understand current risks and concerns, identify potential solutions, and direct future clinical trials and applications.
  • ... The usefulness of adult primary human cardiomyocytes is limited, as these cells contract upon chemical or electrical stimulation only [33]. Despite recent advances in the maturation of iCMs through improved biomechanics and cocultures with other cell types, so far, their maturation status does not reflect adult cardiomyocytes [13,[34][35][36]. Another limitation of our model is the heterogeneity of iCMs. ...
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    Cardiac fibrosis represents a serious clinical problem. Development of novel treatment strategies is currently restricted by the lack of the relevant experimental models in a human genetic context. In this study, we fabricated self-aggregating, scaffold-free, 3D cardiac microtissues using human inducible pluripotent stem cell (iPSC)-derived cardiomyocytes and human cardiac fibroblasts. Fibrotic condition was obtained by treatment of cardiac microtissues with profibrotic cytokine transforming growth factor β1 (TGF-β1), preactivation of foetal cardiac fibroblasts with TGF-β1, or by the use of cardiac fibroblasts obtained from heart failure patients. In our model, TGF-β1 effectively induced profibrotic changes in cardiac fibroblasts and in cardiac microtissues. Fibrotic phenotype of cardiac microtissues was inhibited by treatment with TGF-β-receptor type 1 inhibitor SD208 in a dose-dependent manner. We observed that fibrotic cardiac microtissues substantially increased the spontaneous beating rate by shortening the relaxation phase and showed a lower contraction amplitude. Instead, no changes in action potential profile were detected. Furthermore, we demonstrated that contraction of human cardiac microtissues could be modulated by direct electrical stimulation or treatment with the β-adrenergic receptor agonist isoproterenol. However, in the absence of exogenous agonists, the β-adrenoreceptor blocker nadolol decreased beating rate of fibrotic cardiac microtissues by prolonging relaxation time. Thus, our data suggest that in fibrosis, activated cardiac fibroblasts could promote cardiac contraction rate by a direct stimulation of β-adrenoreceptor signalling. In conclusion, a model of fibrotic cardiac microtissues can be used as a high-throughput model for drug testing and to study cellular and molecular mechanisms of cardiac fibrosis.
  • ... Of note, using the current differentiation protocols, we and other groups have found myofibril force comparable to fetal myofibrils and kinetic properties that more closely reflect those of adult ventricular For this reason, maturation strategies applied to hiPSC-CMs are a primary challenge in the field. In the last few years, many researchers have explored ways to induce greater structure and function development of hiPSC-CMs, using a wide range of methods such as long-term culture (Lundy et al., 2013), biomimetic substrates (Macadangdang et al., 2014(Macadangdang et al., , 2015Ribeiro et al., 2015), electrical or mechanical stimulation (Nunes et al., 2013), co-culture with other cell types (Tulloch et al., 2011), alteration of growth media (Birket et al., 2015) and 3D culture system such as engineered heart tissue (EHTs), heart on a chip or even in vivo neonatal rat heart system (Cho et al., 2017; Figure 3B). Since hiPSC-CMs possess features of developing cardiomyocytes, they can serve as human models for investigating the developmental time-course of early-stage cardiac diseases at cardiac cell level ( Figure 3C). ...
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    Familial dilated cardiomyopathy (DCM) is mostly caused by mutations in genes encoding cytoskeletal and sarcomeric proteins. In the pediatric population, DCM is the predominant type of primitive myocardial disease. A severe form of DCM is associated with mutations in the DMD gene encoding dystrophin, which are the cause of Duchenne Muscular Dystrophy (DMD). DMD-associated cardiomyopathy is still poorly understood and orphan of a specific therapy. In the last 5 years, a rise of interest in disease models using human induced pluripotent stem cells (hiPSCs) has led to more than 50 original studies on DCM models. In this review paper, we provide a comprehensive overview on the advances in DMD cardiomyopathy disease modeling and highlight the most remarkable findings obtained from cardiomyocytes differentiated from hiPSCs of DMD patients. We will also describe how hiPSCs based studies have contributed to the identification of specific myocardial disease mechanisms that may be relevant in the pathogenesis of DCM, representing novel potential therapeutic targets.
  • ... Indeed, a proper modeling of the myocardium should include not only electrical [125,127] but also mechanical [108] stimuli, such as those derived from fluid flow and hydrostatic pressure. Nunes and colleagues described an innovative platform for engineered heart construct in which a 3D cardiac tissue was generated by the self-assembly of hESC-and hiPSC-derived cardiomyocytes seeded into a template PDMS channel, around a surgical suture in type I collagen gels [100]. In this system, named "biowires," cells were subjected to geometric electrical stimulation inducing highly organized cardiac structure and cell maturation. ...
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    In the last decade, the generation of cardiac disease models based on human-induced pluripotent stem cells (hiPSCs) has become of common use, providing new opportunities to overcome the lack of appropriate cardiac models. Although much progress has been made toward the generation of hiPSC-derived cardiomyocytes (hiPS-CMs), several lines of evidence indicate that two-dimensional (2D) cell culturing presents significant limitations, including hiPS-CMs immaturity and the absence of interaction between different cell types and the extracellular matrix. More recently, new advances in bioengineering and co-culture systems have allowed the generation of three-dimensional (3D) constructs based on hiPSC-derived cells. Within these systems, biochemical and physical stimuli influence the maturation of hiPS-CMs, which can show structural and functional properties more similar to those present in adult cardiomyocytes. In this review, we describe the latest advances in 2D- and 3D-hiPSC technology for cardiac disease mechanisms investigation, drug development, and therapeutic studies.
  • ... Several systems have also integrated electrical stimulation into a perfusion bioreactor showing that the electrical stimulation is important in driving cell maturation and allows synchronous contraction of the perfused construct (Radisic et al., 2008). Nunes et al. (2013) generated a platform named biowire by 3D culturing human iPSC (hiPSC) or ESC-CMs together with supporting cells into a template of polydimethylsiloxane (PDMS) channel, around a sterile surgical suture in type I collagen gels. After 1 week, the cells organized along the suture and were electrically stimulated with increasing intensity over time. ...
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    During embryonic morphogenesis, the heart undergoes a complex series of cellular phenotypic maturations (e.g. transition of myocytes from proliferative to quiescent; maturation of the contractile apparatus), and this involves stiffening of the extracellular matrix acting in concert with morphogenetic signals. The maladaptive remodelling of the myocardium, one of the processes involved in determination of heart failure, also involve mechanical cues, with a progressive stiffening of the tissue that produces cellular mechanical damage, inflammation and ultimately myocardial fibrosis. Assessment of mechanical-dependent molecular machinery (in myocardial and non-myocardial cells) is therefore essential to contextualize the maturation of the cardiac tissue at early stages and understand its pathologic evolution in ageing. Since systems to perform multiscale modelling of cellular and tissue mechanics have been evolved, it appears particularly novel to design integrated mechano-molecular models of heart development and disease to be tested in ex vivo reconstituted cells/tissue-mimicking conditions. In the present contribution, we will discuss the latest implication of mechanosensing in heart development and pathology, describe the most recent models of cell/tissue mechanics and delineate novel strategies to target the consequences of heart failure with personalized approaches based on tissue engineering and iPSCs technologies.
  • Article
    The fabrication of biomimetic structures for tissues and organs is emerging in the fields of biomedical engineering and precision medicine. While current progress in biomedical research provides a number of biofabrication methods, the construction of multi-dimensional cardiac tissue is highly challenging due to difficulties in the maturation and synchronization of cardiomyocytes (CMs) in conjunction with other types of cells, such as myofibroblasts and endothelial cells. Here, we show a simple fabrication methodology to construct multi-dimensional cardiac macro tissue (mCMT) by layer-by-layer (LBL) deposition of cells on micro patterned PDMS. mCMTs formed by LBL deposition of pluripotent stem cell (PSC)-derived cardiomyocytes and cardiac fibroblasts formed 3D patterned structures with synchronized beating characteristics. We also demonstrate that cardiac maturation factors such as the gene expression of MLC2v and cTNI and formation of sarcomeres in mCMTs were significantly enhanced by LBL deposition and growth factors during the maturation process. Fabrication of matured mCMTs with synchronized beating enables providing an efficient platform for evaluating the efficacy and toxicity of drug candidates. These results have important implications because mCMTs are applicable to diverse in vitro studies and drug screening methods that require tissue-like structures and functions in a physiological environment.
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    Microphysiological systems, including organoids, 3-D printed tissue constructs and organ-on-a-chips (organ chips), are physiologically relevant in vitro models and have experienced explosive growth in the past decades. Different from conventional, tissue culture plastic-based in vitro models or animal models, microphysiological systems recapitulate key microenvironmental characteristics of human organs and mimic their primary functions. The advent of microphysiological systems is attributed to evolving biomaterials, micro-/nanotechnologies and stem cell biology, which enable the precise control over the matrix properties and the interactions between cells, tissues and organs in physiological conditions. As such, microphysiological systems have been developed to model a broad spectrum of organs from microvasculature, eye, to lung and many others to understand human organ development and disease pathology and facilitate drug discovery. Multiorgans-on-a-chip systems have also been developed by integrating multiple associated organ chips in a single platform, which allows to study and employ the organ function in a systematic approach. Here we first discuss the design principles of microphysiological systems with a focus on the anatomy and physiology of organs, and then review the commonly used fabrication techniques and biomaterials for microphysiological systems. Subsequently, we discuss the recent development of microphysiological systems, and provide our perspectives on advancing microphysiological systems for preclinical investigation and drug discovery of human disease.
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    Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have great potential in biomedical applications. However, the immature state of cardiomyocytes obtained using existing protocols limits the application of hPSC-CMs. Unlike adult cardiac myocytes, hPSC-CMs generate ATP through an immature metabolic pathway-aerobic glycolysis, instead of mitochondrial oxidative phosphorylation (OXPHOS). Hence, metabolic switching is critical for functional maturation in hPSC-CMs. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) is a key regulator of mitochondrial biogenesis and metabolism, which may help promote cardiac maturation during development. In this study, we investigated the effects of PGC-1α and its activator ZLN005 on the maturation of human embryonic stem cell-derived cardiomyocyte (hESC-CM). hESC-CMs were generated using a chemically defined differentiation protocol and supplemented with either ZLN005 or DMSO (control) on differentiating days 10 to 12. Biological assays were then performed around day 30. ZLN005 treatment upregulated the expressions of PGC-1α and mitochondrial function-related genes in hESC-CMs and induced more mature energy metabolism compared with the control group. In addition, ZLN005 treatment increased cell sarcomere length, improved cell calcium handling, and enhanced intercellular connectivity. These findings support an effective approach to promote hESC-CM maturation, which is critical for the application of hESC-CM in disease modeling, drug screening, and engineering cardiac tissue.
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    To accelerate the cardiac drug discovery pipeline, we set out to develop a platform that would be capable of quantifying tissue-level functions such as contractile force and be amenable to standard multiwell-plate manipulations. We report a 96-well-based array of 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues - termed Cardiac MicroRings (CaMiRi) - in custom 3D-print-molded multiwell plates capable of contractile force measurement. Within each well, two elastomeric microcantilevers are situated above a circumferential ramp. The wells are seeded with cell-laden collagen, which, in response to the gradual slope of the circumferential ramp, self-organizes around tip-gated microcantilevers to form contracting CaMiRi. The contractile force exerted by the CaMiRi is measured and calculated using the deflection of the cantilevers. Platform responses were robust and comparable across wells, and we used it to determine an optimal tissue formulation. We validated the contractile force response of CaMiRi using selected cardiotropic compounds with known effects. Additionally, we developed automated protocols for CaMiRi seeding, image acquisition, and analysis to enable the measurement of contractile force with increased throughput. The unique tissue fabrication properties of the platform, and the consequent effects on tissue function, were demonstrated upon adding hPSC-derived epicardial cells to the system. This platform represents an open-source contractile force screening system useful for drug screening and tissue engineering applications.
  • Article
    Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic (hESC) and human induced pluripotent (hiPSC) stem cell-derived cardiomyocytes would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early stage counterparts (PSC-CMs after 20-40 days of in vitro differentiation and culture), late stage hESC-CMs and hiPSC-CMs (80-120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by brightfield microscopy, and a 10-fold increase in the fraction of multinucleated cardiomyocytes. Ultrastructural analysis confirmed improvements in myofibrillar density, alignment, and morphology. Measurement of the contractile performance of late stage hESC-CMs and hiPSC-CMs demonstrated a doubling in shortening magnitude with slowed contraction kinetics compared to early stage cells. We then examined changes in the calcium handling properties of these matured cardiomyocytes and found an increase in calcium release and reuptake rates with no change in maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of key cardiac structural genes including β-MHC and Cx43 in late stage hESC-CMs and hiPSC-CMs. These findings suggest that pluripotent stem cell-derived cardiomyocytes are capable of slowly maturing to more closely resemble the phenotype of adult cardiomyocytes and may eventually possess the potential to regenerate lost myocardium with robust de novo force-producing tissue.
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    New therapies are needed to prevent heart failure after myocardial infarction (MI). As experimental treatment strategies for MI approach translation, safety and efficacy must be established in relevant animal models that mimic the clinical situation. We have developed an injectable hydrogel derived from porcine myocardial extracellular matrix as a scaffold for cardiac repair after MI. We establish the safety and efficacy of this injectable biomaterial in large- and small-animal studies that simulate the clinical setting. Infarcted pigs were treated with percutaneous transendocardial injections of the myocardial matrix hydrogel 2 weeks after MI and evaluated after 3 months. Echocardiography indicated improvement in cardiac function, ventricular volumes, and global wall motion scores. Furthermore, a significantly larger zone of cardiac muscle was found at the endocardium in matrix-injected pigs compared to controls. In rats, we establish the safety of this biomaterial and explore the host response via direct injection into the left ventricular lumen and in an inflammation study, both of which support the biocompatibility of this material. Hemocompatibility studies with human blood indicate that exposure to the material at relevant concentrations does not affect clotting times or platelet activation. This work therefore provides a strong platform to move forward in clinical studies with this cardiac-specific biomaterial that can be delivered by catheter.
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    Human embryonic stem cells have emerged as the prototypical source from which cardiomyocytes can be derived for use in drug discovery and cell therapy. However, such applications require that these cardiomyocytes (hESC-CMs) faithfully recapitulate the physiology of adult cells, especially in relation to their electrophysiological and contractile function. We review what is known about the electrophysiology of hESC-CMs in terms of beating rate, action potential characteristics, ionic currents, and cellular coupling as well as their contractility in terms of calcium cycling and contraction. We also discuss the heterogeneity in cellular phenotypes that arises from variability in cardiac differentiation, maturation, and culture conditions, and summarize present strategies that have been implemented to reduce this heterogeneity. Finally, we present original electrophysiological data from optical maps of hESC-CM clusters.
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    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl(2) (n = 22). Patch-clamp techniques were used to record I(Kr) and I(K1) from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p
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    Human pluripotent stem cell (hPSC-) derived cardiomyocytes have potential applications in drug discovery, toxicity testing, developmental studies, and regenerative medicine. Before these cells can be reliably utilized, characterization of their functionality is required to establish their similarity to native cardiomyocytes. We tracked fluorescent beads embedded in 4.4-99.7 kPa polyacrylamide hydrogels beneath contracting neonatal rat cardiomyocytes and cardiomyocytes generated from hPSCs via growth-factor-induced directed differentiation to measure contractile output in response to changes in substrate mechanics. Contraction stress was determined using traction force microscopy, and morphology was characterized by immunocytochemistry for α-actinin and subsequent image analysis. We found that contraction stress of all types of cardiomyocytes increased with substrate stiffness. This effect was not linked to beating rate or morphology. We demonstrated that hPSC-derived cardiomyocyte contractility responded appropriately to isoprenaline and remained stable in culture over a period of 2 months. This study demonstrates that hPSC-derived cardiomyocytes have appropriate functional responses to substrate stiffness and to a pharmaceutical agent, which motivates their use in further applications such as drug evaluation and cardiac therapies.
  • Article
    An inwardly rectifying potassium channel (Kir) is a kind of protein complex that is widely expressed on excitable and nonexcitable cell membranes. Kir channels serve important roles in cellular physiology such as cell excitability and K+ homeostasis. The Kirs (KIR1-7) are regulated by many factors: phosphatidylinosital-4, 5-bisphosphate (PIP2), ATP, or G-proteins. Other factors like polyamines, kinases, pH, and Na+ ions act cooperatively to modulate Kir channels. Different types and specific distributions of KIR channels determine the diversity of regulatory mechanisms. This review provides insight into Kir channel regulation.
  • Article
    During neonatal development, there is an increase in myocardial stiffness that coincides with an increase in the contractility of the heart. In vitro assays have shown that substrate stiffness plays a role in regulating the twitch forces produced by immature cardiomyocytes. However, its effect on twitch power is unclear due to difficulties in measuring the twitch velocity of cardiomyocytes. Here, we introduce what we consider a novel approach to quantify twitch power by combining the temporal resolution of optical line scanning with the subcellular force resolution of micropost arrays. Using this approach, twitch power was found to be greater for cells cultured on stiffer posts, despite having lower twitch velocities. The increased power was attributed in part to improved myofibril structure (increased sarcomere length and Z-band width) and intracellular calcium levels. Immunofluorescent staining of α-actin revealed that cardiomyocytes had greater sarcomere length and Z-band width when cultured on stiffer arrays. Moreover, the concentration of intracellular calcium at rest and its rise with each twitch contraction was greater for cells on the stiffer posts. Altogether, these findings indicate that cardiomyocytes respond to substrate stiffness with biomechanical and biochemical changes that lead to an increase in cardiac contractility.