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Bioengineering Approaches to Mature Human Pluripotent Stem Cell-Derived Cardiomyocytes


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Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) represent a potential unlimited cell supply for cardiac tissue engineering and possibly regenerative medicine applications. However, hPSC-CMs produced by current protocols are not representative of native adult human cardiomyocytes as they display immature gene expression profile, structure and function. In order to improve hPSC-CM maturity and function, various approaches have been developed, including genetic manipulations to induce gene expression, delivery of biochemical factors, such as triiodothyronine and alpha-adrenergic agonist phenylephrine, induction of cell alignment in 3D tissues, mechanical stress as a mimic of cardiac load and electrical stimulation/pacing or a combination of these. In this mini review, we discuss biomimetic strategies for the maturation for hPSC-CMs with a particular focus on electromechanical conditioning methods.
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published: 09 March 2017
doi: 10.3389/fcell.2017.00019
Frontiers in Cell and Developmental Biology | 1March 2017 | Volume 5 | Article 19
Edited by:
Cedric Viero,
Servier, France
Reviewed by:
Philippe Bourin,
Univercell Biosolutions, France
Tomo Saric,
University of Cologne, Germany
Elena Matsa,
Stanford University, USA
Joao Mario Martins Bigares,
Wales Heart Research Institute-School
of Medicine-Cardiff University, UK
Sara S. Nunes
Specialty section:
This article was submitted to
Stem Cell Research,
a section of the journal
Frontiers in Cell and Developmental
Received: 01 December 2016
Accepted: 21 February 2017
Published: 09 March 2017
Sun X and Nunes SS (2017)
Bioengineering Approaches to Mature
Human Pluripotent Stem Cell-Derived
Front. Cell Dev. Biol. 5:19.
doi: 10.3389/fcell.2017.00019
Bioengineering Approaches to
Mature Human Pluripotent Stem
Cell-Derived Cardiomyocytes
Xuetao Sun 1and Sara S. Nunes 1, 2, 3*
1Toronto General Research Institute, University Health Network, Toronto, ON, Canada, 2Institute of Biomaterials and
Biomedical Engineering, University of Toronto, Toronto, ON, Canada, 3Heart & Stroke/Richard Lewar Centre of Excellence,
University of Toronto, Toronto, ON, Canada
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) represent a potential
unlimited cell supply for cardiac tissue engineering and possibly regenerative medicine
applications. However, hPSC-CMs produced by current protocols are not representative
of native adult human cardiomyocytes as they display immature gene expression
profile, structure and function. In order to improve hPSC-CM maturity and function,
various approaches have been developed, including genetic manipulations to induce
gene expression, delivery of biochemical factors, such as triiodothyronine and
alpha-adrenergic agonist phenylephrine, induction of cell alignment in 3D tissues,
mechanical stress as a mimic of cardiac load and electrical stimulation/pacing or a
combination of these. In this mini review, we discuss biomimetic strategies for the
maturation for hPSC-CMs with a particular focus on electromechanical conditioning
Keywords: cardiomyocytes, cardiac regeneration, stem cell, biomaterials, cell therapy, electrical stimulation,
mechanical stimulation
Human embryonic stem cells (hESCs), first isolated from inner cell mass of blastocysts, possess
the capacity to differentiate into cells of all three germ layers (Thomson et al., 1998). Similar
characteristics can also be found in human induced pluripotent stem cells (hiPSCs), which are
generated from terminally differentiated, adult cells by genetically reprogramming via expression
of a set of transcription factors (Takahashi et al., 2007; Yu et al., 2007). These cells circumvent the
ethical concerns associated with hESCs and allow a potential autologous approach without the need
for long-term immunosuppression. Cardiomyocytes can be differentiated from both hESCs and
hiPSCs using directed differentiation approaches, which are based on the stage-specific treatment
with cardiogenic-inducing signaling factors (Laflamme et al., 2007; Yang et al., 2008).
However, human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) (including hESC-
CM and hiPSC-CM) display immature characteristics when compared to adult cardiomyocytes,
such as (Table 1):
1) Genetically, hPSC-CMs express much lower levels of cardiac contractile and cytoskeletal genes
(Cao et al., 2008; Xu et al., 2009). Early hPSC-CMs have high proliferation rates (Robertson
et al., 2013) while adult cardiomyocytes are considered non-proliferative (0.5% proliferation
per year) (Bergmann et al., 2009).
2) Morphologically, hPSC-CMs are small, disorganized, mononucleated, round/triangular in
shape; while adult human cardiomyocytes are large, highly organized, 25% binucleated
Sun and Nunes Bioengineering Pro-maturation Strategies for hPSC-CMs
TABLE 1 | Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) vs. adult ventricular cardiomyocytes.
Criteria hPSC-CM Adult ventricular cardiomyocytes References
Structure Shape Round Rod Gerdes et al., 1992; Lundy et al., 2013
Cell surface area 10212–14418 µm2500–1294 µm2Li et al., 1996; Lundy et al., 2013; Ribeiro et al.,
Gene Expression MYH7 <MYH6 TNNI3 <TNNI1 MYH7 >MYH6 TNNI3 >TNNI1 Xu et al., 2009
Nuclei Mononuclear 25% binucleation Olivetti et al., 1996; Snir et al., 2003
Sarcomere 1.65 µm2.2 µmVan Der Velden et al., 1998; Lundy et al., 2013
T-tubules Absent Present Brette and Orchard, 2003; Yang et al., 2014a
Energy and force Mitochondria Near nuclei, small fraction Throughout cell; 20–40% of cell volume Schaper et al., 1985; Gherghiceanu et al., 2011
Energy Glycolysis ß-oxidation of fatty acid Lopaschuk and Jaswal, 2010; Kim et al., 2013
Contractile force 0.22 ±0.70 to 11.8 ±4.5 mN/mm251 ±8 mN/mm2Van Der Velden et al., 1998; Kita-Matsuo et al.,
2009; Zhang et al., 2013
Proliferation Early hPSC-CM: Yes Late hPSC-CM: No Considered non-proliferative Bergmann et al., 2009; Robertson et al., 2013
Calcium transients Inefficient Efficient Itzhaki et al., 2011
Slow Fast Yang et al., 2014a
AP(action potential) properties Upstroke velocity 15–50 V/s 180–400 V/s Dangman et al., 1982; Drouin et al., 1995; He
et al., 2003; Lundy et al., 2013
Resting membrane potential 20 to 60 mV 90 mV Drouin et al., 1995; Mummery et al., 2003;
Lundy et al., 2013
Conduction velocity 2.1–20 cm/s 41–84 cm/s Nanthakumar et al., 2007; Caspi et al., 2009;
Lee et al., 2012
Capacitance 5–30 pF 150 pF Drouin et al., 1995; Blazeski et al., 2012
Automaticity Spontaneous beating Quiescent Chen et al., 2009; Lundy et al., 2013
mRNA level Cav1.2 Similar to adult cardiomyocyte Satin et al., 2008
Cavß1 20 fold lower than adult cardiomyocyte Satin et al., 2008
RyR2 1000 fold lower than adult cardiomyocyte Satin et al., 2008
Ion channel density (pA/pF) INa 20 to 330 ∼ −50 Valdivia et al., 2005; Fatima et al., 2013;
Ivashchenko et al., 2013
ICaL 2.2 to 11 2.3 to ∼ −10 Magyar et al., 2000; Er et al., 2009; Fu et al.,
2010; Otsuji et al., 2010
Ito 2.5–13.7 2.3–9.2 Beuckelmann et al., 1993; Wettwer et al.,
1994; Ma et al., 2011; Cordeiro et al., 2013
IKs 0.3–0.7 0.18 Virag et al., 2001; Otsuji et al., 2010; Ma et al.,
2011; Jonsson et al., 2012
IKr 0.4–0.8 0.6 Jost et al., 2009; Fu et al., 2011; Ma et al.,
IK10.6 to 3.4 ∼ −12 Schram et al., 2003; Sartiani et al., 2007
INCX 3.6–7.9 (Ca2+inward mode) 2.5–3 Weber et al., 2003; Fu et al., 2010
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Sun and Nunes Bioengineering Pro-maturation Strategies for hPSC-CMs
(Olivetti et al., 1996) with rod-like shape. In addition, hPSC-
CMs possess sparse, disorganized and shorter sarcomeres
(1.6 µm), and few or no transverse tubules (T-tubules).
Normal adult cardiomyocytes exhibit well-aligned, longer
sarcomeres (2.2 µm) characterized by the presence of Z
discs, and I-, H-, A-, and M-bands.
3) Metabolically, hPSC-CMs are characterized by a relatively low
number of mitochondria and a dependence on glycolysis as
opposed to a predominantly fatty acid metabolism in adult
cardiomyocytes (Yang et al., 2014a).
4) Functionally, hPSC-CMs display a force-generation capacity
(0.22 ±0.70 mN/mm211.8 ±4.5 mN/mm2) (Kita-
Matsuo et al., 2009; Zhang et al., 2013) comparable to fetal
cardiomyocytes (2nd trimester) (0.4 mN/mm2) (Ribeiro
et al., 2015) and much lower than adult (51 mN/mm2) (Van
Der Velden et al., 1998).
5) Electrophysiologically, hPSC-CMs show greater
heterogeneity and immaturity in their electrical properties
than adult cardiomyocytes including: (a) reduced electrical
excitability; (b) decreased excitation–contraction coupling
(ECC); (c) higher resting membrane potential (20 to 60
mV vs. ∼ −90 mV); (d) low capacitance; (e) smaller upstroke
(15–50 vs. 180–400 V/s) and conduction velocity (2.1–20 vs.
41–84 cm/s); and (f) presence of automaticity (spontaneous
beating), which is found in early fetal cardiomyocytes and
later specific to pacemaker cells.
These immature features may limit hPSC-CM application and
highlight the need for the development of pro-maturation
strategies to obtain human adult cardiomyocytes in vitro. Given
the complexity of the cardiomyocyte structure and function, the
term “maturation” represents multi-faceted properties used to
evaluate their maturation state. However, the properties reported
in different studies have often varied (Figure 1) making it difficult
to draw a direct comparison.
Cardiomyocytes undergo a series of structural changes and
ultimately reach full maturity in the adult heart, which enables
them to fulfill their functional role. This development process is
long (years) and under complex regulation (Ahuja et al., 2007).
hPSC-CMs could mature to adult-like size and morphology
within 3 months post-transplantation into infarcted hearts
of non-human primates (Chong et al., 2014). Long-term
culture in vitro (80–120 days) has been suggested effective in
improving the maturity of hPSC-CMs (Lundy et al., 2013).
However, this is very time-consuming and cost prohibitive.
More strategies to promote the maturity of hPSC-CMs include:
genetic manipulation (e.g., adenovirus-mediated overexpressing
of Kir2.1 Lieu et al., 2013), modulation of microRNAs (e.g.,
lentivirus-mediated overexpression of miR-1 Fu et al., 2011),
delivery of biochemical factors, such as triiodothyronine (Yang
et al., 2014b) and alpha-adrenergic agonist phenylephrine (Foldes
et al., 2011), induction of cell alignment in 3D tissues (Zhu et al.,
2014), and electrical and/or mechanical stimulation.
Of these, mechanical and electrical stimulation are major
biophysical cues that play critical roles in cardiomyocyte growth
and maturation during cardiac development and have been tested
as maturation cues for hPSC-CM. To replicate electromechanical
forces in vitro, hPSC-CMs are cultured in a biomimetic
environment comparable to native cardiac microarchitecture and
subjected to mechanical and/or electrical stimuli. The goal is
to promote the maturity of hPSC-CMs while improving our
understanding of the mechanisms responsible for the adaptive
changes of cardiac tissue under physiological and pathological
Mechanical Stress
Mechanical force plays a critical role during development of
cardiac structure and function (Zimmermann, 2013). It may thus
be important to consider the presence of proper mechanical
signaling or cues when designing a platform for the maturation
of hPSC-CMs, regardless of whether it is in 2D or 3D. Mechanical
stimulation on cells can be implemented by adjusting the
substrate properties (stiffness/topography) and/or stretching.
These have been suggested to be effective in improving the
maturation properties of hPSC-CMs.
The effect of substrate rigidity on maturation can be
demonstrated by plating spontaneously contracting hPSC-CMs
on extracellular matrix (ECM) protein-coated tissue culture
surfaces where the matrix composition can be altered to
obtain physiological range of substrate stiffness. It’s been shown
that in a range of 4–80 kPa polyacrylamide hydrogels, the
highest differentiation efficiency using hESCs was achieved at
50 kPa (Hazeltine et al., 2014), and that contractile output
of cardiomyocytes increased in response to increased substrate
stiffness (4.4–99.7 kPa) (Hazeltine et al., 2012). Two-dimensional
substrates can also be micropatterned to improve hPSC-CM
alignment and sarcoplasmic reticulum (SR) Ca2+cycling (Rao
et al., 2013; Salick et al., 2014), which suggest improved
maturation. However, these two-dimensional structures lack
important features of the natural 3D environment that affect the
cell biology.
Stretch is the major method used to deliver mechanical
stimuli to hPSC-CMs and generally done by applying external
mechanical stress to hPSC-CM constructs in a static (achieved
by increasing the stretch over time or directly to a fixed distance)
or dynamic (mimicking the native cyclic mechanical stimulus on
the cardiac muscle) fashion.
Early studies to test the effect of mechanical stress on
immature cardiomyocytes were performed by seeding cells
in collagen/Matrigel matrix, casting it in circular molds and,
following tissue compaction, the engineered heart tissues (EHT)
(Zimmermann et al., 2002) were subjected to uniaxial cyclic
stretch (2 Hz, 10% elongation). After 1 week, EHTs displayed
important hallmarks of mature myocardium: organized muscle
bundles with aligned sarcomeres and positive force-frequency
relationship (Endoh, 2004). Furthermore, these hEHTs show
a positive inotropic response to extracellular Ca2+and
isoproterenol (Streckfuss-Bomeke et al., 2013).
In another study, hEHTs were generated by mixing single-
cell hESC-CMs in a fibrin/Matrigel gel and casting into a
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Sun and Nunes Bioengineering Pro-maturation Strategies for hPSC-CMs
FIGURE 1 | Schematic diagram illustrating the strategies to promote and the assessment of the maturation of human pluripotent stem cell
(hPSC)-derived cardiomyocytes (hPSC-CM). These approaches may be used individually or in any combination to promote hPSC-CM maturation. The
assessment of the maturation should be physiologically relevant, including readout from morphology (cell alignment, cell shape/size, sarcomeres and T-tubules), gene
expression (sarcomeric, ion channels and their regulators), and function (calcium handling, ECC, electrophysiology, contraction and transplantation).
12 ×3×3 mm agarose mold in which two elastic silicone
posts were inserted from above (Schaaf et al., 2011). Upon
compaction, the cardiac construct strip anchored to the posts
was subjected to static strain and displayed improved cell
alignment and sarcomeric organization compared with age-
matched EBs, and expressed connexin-43 but not in intercalated
disks. Transcription levels of β-MHC increased significantly
over time in hEHTs but not in EBs. The hEHTs demonstrated
contractions 5–10 days after casting, reached regular (mean
0.5 Hz) and strong (mean 100 mN) contractions for up to
8 weeks. The constructs exhibited positive chronotropic and
inotropic response to increasing concentrations of extracellular
Ca2+(Schaaf et al., 2011).
Cardiac constructs were also generated by casting collagen-
based hPSC-CMs gels in a 20 mm ×3 mm channel, in which
the ends of the construct were anchored into nylon mesh tabs
attached to a deformable silicon floor of the well (tissue train,
Flexcell). Upon cell remodeling and gel contraction, the cardiac
constructs were held by the nylon tabs under static tension
or subjected to controlled cyclic stress (1 Hz, 5% elongation)
(Tulloch et al., 2011). After 4 days, there was improvement in cell
alignment and striations within the constructs. Cyclic stretch also
upregulated transcripts of β-MHC, cTnT, ANP, BNP, CACNA1C,
RYR2, and SERCA2 (Tulloch et al., 2011). Functionally, cardiac
constructs subjected to 3 weeks of static strain have increased
their active force in response to increased resting length (Tulloch
et al., 2011), analogous to Frank-Starling curves (an increase
in force with increased preload known as length-dependent
activation) (Glower et al., 1985).
Mihic et al. (2014) used cyclic mechanical stretch to
enhance the viability and functional maturation of hPSC-
CM tissue constructs prior to implantation into the damaged
myocardium. The constructs were generated by seeding hESC-
CMs in a 30 ×10 ×7 mm gelatin sponges. After 2 days of
compaction, the cardiac constructs were subjected to 3 days
of uniaxial cyclic stretch (1.25 Hz, 12% elongation). Compared
to unstretched controls, cyclically stretched cardiac constructs
exhibited increased number of cells, cell size and elongation,
increased expression of connexin-43, and upregulated mRNA
expression of MYH7, CACNA1C, HCN4, KCNH2, SCN5A, and
KCNJ2. Functionally, the cyclically stretched cardiac constructs
were demonstrated faster contraction rates with shorter calcium
cycle duration.
Zhang et al. (2013) used a platform to promote hESC-
CMs alignment within cardiac patch via locally controlling the
direction of passive tension. hESC-CMs (48–90% purity) were
cultured for 2 weeks in a mixture of fibrin and Matrigel in 7
×7 mm2polydimethylsiloxane (PDMS) molds with staggered
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Sun and Nunes Bioengineering Pro-maturation Strategies for hPSC-CMs
hexagonal posts (1.2 mm long) to generate a cardiac patches with
elliptical pores formed around the posts upon tissue compaction.
The resultant hESC-CMs in the 3D patches exhibited a maximal
conduction velocity of 25.1 cm/s, and longer sarcomeres (2.09
±0.02 vs. 1.77 ±0.01 µm), and enhanced expression of
genes involved in cardiac contractile function, including cTnT,
αMHC, CASQ2 and SERCA2 when compared to age and purity
matched hESC-CMs cultured in monolayers (Zhang et al., 2013).
Moreover, maximum contractile forces and active stresses of
cardiac patches were 3.0 ±1.1 mN and 11.8 ±4.5 mN/mm2,
respectively, and the patches were shown to generate Frank-
Starling curves with respect to both active and passive force
as well as positive inotropic response to isoproterenol (Zhang
et al., 2013). These author’s findings highlight the superiority of
3D vs. 2D culture models. However, no improvements in the
electrophysiological properties were reported.
These studies have established the significance of mechanical
stimuli as a maturation cue for hPSC-CMs. However, it
should be noted that the contractile forces measured from
the aforementioned EHTs were related to the biomaterial
composition (e.g., collagen vs. fibrin). Such material variability
may affect the hPSC-CM phenotype, which consequently cause
the variation of functional readout including contractile force.
Furthermore, other variables in these mechanical stimulation
regimes, such as the cell culture condition and duration
of stimulation, makes it difficult to determine an optimal
mechanical stress protocol for generating mature cardiac tissues.
Electrical Stimulation
Cardiomyocytes are rhythmically and synchronously contracting
in response to electrical signals. This process of converting
electrical signals into contraction (commonly known
as excitation-contraction coupling or ECC) requires the
coordinated activity of several ion channels (Liu et al., 2016).
The developmental changes in these ion channels are under
complex regulation and accompany changes in electrical
properties of cardiomyocytes across the fetal and postnatal
stages, with a specific electrophysiological “signature” in mature
adult cardiomyocytes. hPSC-CMs have been shown to be
electrophysiologically immature. Studies recapitulating in vitro
the electrical activity cardiomyocytes are exposed to in vivo have
demonstrated that electrical stimulation promotes aspects of
hPSC-CM maturation.
We have devised a platform called “biowire,” to mature hPSC-
CMs by combining 3D culture and electrical stimulation (Nunes
et al., 2013; Sun and Nunes, 2016). Biowires were generated by
culturing hPSC-CMs in collagen hydrogels around a surgical
suture to form cardiac tissues of 600 µm in diameter (Nunes
et al., 2013). Biowires were subjected to 7 days of electrical field
stimulation (3 V/cm, 1 ms pulse, starting at 1 Hz with step-wise
increases to 3 or 6 Hz). At the endpoint, hPSC-CMs exhibited
properties compatible with cardiomyocyte maturation, such as
improved cell and myofibril alignment, improved sarcomeric
banding, larger cardiomyocyte area and lower proliferation rates,
compared with age-matched EBs. Automaticity was significantly
higher in EB-derived cardiomyocytes compared to control
biowires, which was comparable to that in biowires subjected
to the 6-Hz regimen. Electrical stimulation also significantly
increased the conduction velocity of biowires from 11.5 to
18.5 cm/s. Biowires exposed to electrical stimulation also showed
increased Ca2+transient amplitudes vs. unstimulated controls.
hPSC-CMs in biowires exhibited improved hERG current and
inward rectifier current (Ik1) densities, which were further
enhanced by electrical stimulation. This study revealed for the
first time that these changes were dependent on the electrical
stimulation rate as evidenced by greater extent of maturation
obtained in the biowires exposed to the 6 Hz stimulation
ramp-up regimen (vs 3 Hz) (Nunes et al., 2013). However,
given the presence of the silk suture the force of contraction
generated by the hPSC-CMs could not be measured. The use of a
biodegradable suture may make this possible in the future.
Others have shown that hESC-CMs subjected to 2-week-long
electrical conditioning (2.5 V/cm, 1 Hz, 5 ms pulse) exhibited
lower spontaneous activity, hyperpolarized resting potential,
increased intracellular Ca2+transients, structured organization
of myofilaments, and an upregulation of Kir2.1, CSQ2, junctin,
triadin, SERCA, Cav3, Amp2, MHC, and MLC genes (Lieu
et al., 2013). In another study, beating EBs seeded on gelatin-
coated plates and subjected to 4-day-long electrical stimulation
(6.6 V/cm, 1 Hz, 2 ms pulse) exhibited cell elongation,
increased action potential duration, increased Ca2+transients
and increased expression of cardiac-specific gene including
HCN1, MLC2V, SCN5A, SERCA, Kv4.3, and GATA4 (Chan et al.,
In a recent study, EBs differentiated from hPSCs were
subjected to electrical conditioning (5 V/cm, 0.5, 1 and 2
Hz, 2 ms pulse) continuously for 7 days (Eng et al., 2016).
Such electrical stimulation enhanced connexin expression and
sarcomeric structure. Cardiomyocytes adapted their autonomous
beating rate to the frequency at which they were stimulated, an
effect mediated by the emergence of a rapidly depolarizing cell
type, and the expression of hERG. The resultant cardiomyocytes
were robust and could maintain the adapted beating rates for up
to 2 weeks after the cessation of electrical stimulation (Eng et al.,
While electrical stimulation has consistently improved the
maturation of hPSC-CMs, one possible drawback of utilizing
electrical stimulation is the limited scalability. This may not be
of concern for its utilization in drug screening platforms but
may hinder its application in cell maturation for regenerative
medicine applications.
Combined Mechanical and Electrical
Efforts have also been made to examine the effect of combining
mechanical and electrical stimulation, sequentially or
concurrently, to hPSC-CM constructs. Hirt et al. (2014)
generated spontaneously beating fibrin/Matrigel-based hPSC-
CM constructs with static stretch and subjected them to electrical
field stimulation (2 V/cm, 4 ms pulse, 2 Hz for 1 week and
1.5 Hz thereafter) for at least 10 days. This increased cell
alignment, sarcomere organization, Ca2+-response curves,
force generation and inotropic response to β-adrenergic
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Sun and Nunes Bioengineering Pro-maturation Strategies for hPSC-CMs
stimulation while decreasing automaticity. (Hirt et al.,
In another study, hPSC-CMs were embedded into a collagen-
based scaffold and then subjected to static stress for 2 or 1 week
of static stress and 1 week of combined static stress and electrical
pacing (5 V/cm, 2 Hz, 5 ms pulse) (Ruan et al., 2016). Compared
to no stress/no pacing controls, 2-week static stress conditioning
promoted cell alignment, passive stiffness, cardiac hypertrophy,
and increased contractility of hPSC-CM constructs (0.63 ±0.10
mN/mm2vs. 0.055 ±0.009 mN/mm2). The contractility of the
constructs could be further increased by combining stretch with
1-week electrical stimulation (1.34 ±0.19 mN/mm2). Combined
static stress and electrical stimulation enhanced expression of
SR-related proteins (RYR2 and SERCA2) (Ruan et al., 2016).
The efforts to mimic native biophysical stimulation to mature
hPSC-CMs have led to a number of effective strategies to
mature hPSC-CMs and advance our understanding of how
these cues affect cardiomyocyte structure and function. However,
the properties assessed often varied between studies making
it difficult to draw a direct comparison between the different
strategies. This is accentuated by the lack of uniformity in
cardiomyocyte maturation in artificial, in vitro settings where
electrical stimulation seems to have a stronger impact on
electrical properties while mechanical stimulation improves
structural components and force generation with smaller impact
on electrical properties. This argues for a homogeneity in the
parameters utilized as functional readouts (electrophysiology,
calcium dynamics, force of contraction and ultrastructure).
Although progress has been made, an adult-like phenotype in
vitro has yet to be reported. This can have multiple limitations
regarding application. First, the maturation status of hPSC-CMs
should be staged and documented depending on the potential
application sceneries. For example, for myocardial infarction
(MI) therapy, less mature cardiomyocytes might adapt better for
transplantation into the infarcted myocardium (Reinecke et al.,
1999). However, the best-defined maturation stage of hPSC-CMs
for transplantation into MI remains to be determined.
Second, the hPSC-CMs obtained from existing cardiac
differentiation protocols are a mixed population of ventricular-,
atrial-, and nodal-like cells. Such heterogeneity represents
a limitation for certain applications, e.g., transplantation of
high purity of ventricular cardiomyocytes to potentially avoid
tachyarrhythmias caused by spontaneously firing (nodal-like)
cells; and high throughput (HTS) drug testing platforms for
cardiac drug responses.
Third, the significance of the in vivo environment for the
maturation of cardiomyocytes should be noted. Immature hPSC-
CMs differentiated in vitro could mature to adult size and
morphology after transplantation into the infarcted hearts of
non-human primates (Chong et al., 2014).
Proper cardiac development and function requires other
cell types, such as fibroblasts, endothelial, and smooth muscle
cells that may have an impact in cardiomyocyte maturation.
While there is still controversy regarding whether non-
cardiomyocytes may promote hPSC-CM maturation via
secretion of undefined factors (Kim et al., 2010; Lundy et al.,
2013), a full understanding of these interactions may help
to uncover unknown cues, which could then be used to
promote hPSC-CM maturation in the absence of a specific
cell type.
hPSC-CMs have shown great promises in various applications
including cardiac development, regenerative medicine, disease
modeling, and drug testing/screening/discovery. The generation
of a large number of mature hPSC-CMs is essential to achieve
these goals. Importantly, these approaches are not mutually
exclusive (Figure 1) and there’s been a trend to combine
the existing strategies to obtain more effective maturation.
The combination of mechanical and electrical stimulation has
shown possible synergistic effects with a 2-fold increase in
contractility (Ruan et al., 2016). This trend should lead to exciting
discoveries regarding hPSC-CM maturation and possibly the
achievement of adult-like cardiomyocytes in vitro for the first
XS and SN conceived and wrote the manuscript.
This work was supported by a grant-in-aid from the Heart and
Stroke Foundation of Canada (G-14-0006265), operating grants
from the Canadian Institutes of Health Research (137352 and
143066) and a J.P. Bickell foundation grant (1013821) to SN.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2017 Sun and Nunes. This is an open-access article distributed under the
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reproduction in other forums is permitted, provided the original author(s) or licensor
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Frontiers in Cell and Developmental Biology | 8March 2017 | Volume 5 | Article 19
... Maturation of the hiPSC-CMs remains one of the most significant barriers to their application in research and clinical therapeutics. While they are still able to recapitulate many arrhythmogenic phenotypes observed in patients and other models (e.g., murine models, biochemical studies), hiPSC-CMs produced under standard differentiation protocols retain many of the structural and functional qualities of a fetal In addition, the growth of hiPSC-CMs as 3D tissue, such as organoids, spheroids, or microtissue mounted on structural scaffolding, has helped to enhance some phenotypes that are missing in immature 2D tissue, including the development of T-tubules (TT) for excitation-contraction coupling (Kim et al. 2010a;Parikh et al. 2017;Ronaldson-Bouchard et al. 2018;Sun and Nunes 2017). Integration of these approaches, which involve mechanochemical and metabolic cues, could help activate multiple, cascading molecular signalling pathways to enact developmental changes in the cells ranging from transcriptional and protein expression to tissue and organ morphology and function (Fig. 2). ...
... Following population purification, chemical, electromechanical, and/or structural treatments are used to induce maturation to produce hiPSC-CMs with more adult-like phenotypes cardiomyocyte. Current research is focused on tackling the challenge of simulating the cardiomyocyte maturation process in vitro(Karbassi et al. 2020;Lundy et al. 2013;Marchianò et al. 2019;Piccini et al. 2015;Sun and Nunes 2017), though complicated and not fully achieved yet. Many strategies for hiPSC-CM maturation are outlined in the literature and encompass guided electrical, chemical, and metabolic treatments(Feyen et al. 2020;Garbern et al. 2020;Ronaldson-Bouchard et al. 2018;Sun and Nunes 2017). ...
... Current research is focused on tackling the challenge of simulating the cardiomyocyte maturation process in vitro(Karbassi et al. 2020;Lundy et al. 2013;Marchianò et al. 2019;Piccini et al. 2015;Sun and Nunes 2017), though complicated and not fully achieved yet. Many strategies for hiPSC-CM maturation are outlined in the literature and encompass guided electrical, chemical, and metabolic treatments(Feyen et al. 2020;Garbern et al. 2020;Ronaldson-Bouchard et al. 2018;Sun and Nunes 2017). ...
Full-text available
Cardiac arrhythmias can arise due to a host of both genetic and acquired factors. Specifically, the genetic basis of arrhythmogenesis is not fully understood due to the lack of robust models that reliably recapitulate human physiology. Human- induced pluripotent stem cells (hiPSCs) have strengthened regenerative medicine by producing cells that bear the genetic signature of patients being studied. Upon differentiation into hiPSC-derived cardiomyocytes (hiPSC-CMs), these cells can be used to phenotype known mutations or suspected variants that may contribute to abnormal electrical activity in the heart. Furthermore, novel therapeutics can be screened for the management and treatment of arrhythmias in patient-specific hiPSC-CMs. In this chapter, we will briefly discuss the practical utility of hiPSC- CMs to study inherited arrhythmias with a specific focus on atrial fibrillation (AF), catecholaminergic polymorphic ventricular tachycardia (CPVT), and dis- ruptive electrical events that may occur in patients with hypertrophic cardiomy- opathy (HCM). We will describe an investigative pipeline that integrates genome editing, tissue engineering, biobanking, and systems biology as complementary approaches. Together, these various applications are directed toward a common goal of bench-to-bedside characterization of arrhythmias in patient-specific hiPSC-CMs.
... Multiple pieces of evidence have demonstrated that the maturation of stem cells into fully differentiated CMs is greatly improved by the application of mechanical forces. Currently, most of the efforts have been placed on applying distinct types of mechanical stress to different cell types, alone or in combination with distinct biocompatible scaffolds [247][248][249], with the aim of generating cardiac patches that might serve as therapeutic tools to heal the injured heart. Efforts have been made modeling distinct CMs derived from different sources, such as mesenchymal stem cells [250][251][252], cardiomyocyte progenitor cells [253][254][255][256], a mixture of cardiac cells, embryonic [257] and adult [258] CMs, induced pluripotent stem cells-derived CMs [259] and embryonic stem cell-derived CMs [260,261]. ...
Full-text available
Cardiovascular diseases are the leading cause of death worldwide, among which ischemic heart disease is the most representative. Myocardial infarction results from occlusion of a coronary artery, which leads to an insufficient blood supply to the myocardium. As it is well known, the massive loss of cardiomyocytes cannot be solved due the limited regenerative ability of the adult mammalian hearts. In contrast, some lower vertebrate species can regenerate the heart after an injury; their study has disclosed some of the involved cell types, molecular mechanisms and signaling pathways during the regenerative process. In this ‘two parts’ review, we discuss the current state-of-the-art of the main response to achieve heart regeneration, where several processes are involved and essential for cardiac regeneration.
... Roller electrospinning and far-field electrospinning are the most effective methods to manufacture aligned and random fiber scaffolds [40], which can acquire superior morphology and mechanical properties of fibers, as shown in SEM micrographs (Figure 2a,b). Fiber density is an important factor that affects cell seeding efficiency. ...
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Electrospun nanofiber constructs represent a promising alternative for mimicking the natural extracellular matrix in vitro and have significant potential for cardiac patch applications. While the effect of fiber orientation on the morphological structure of cardiomyocytes has been investigated, fibers only provide contact guidance without accounting for substrate stiffness due to their deposition on rigid substrates (e.g., glass or polystyrene). This paper introduces an in situ fabrication method for suspended and well aligned nanofibrous scaffolds via roller electrospinning, providing an anisotropic microenvironment with reduced stiffness for cardiac tissue engineering. A fiber surface modification strategy, utilizing oxygen plasma treatment combined with sodium dodecyl sulfate solution, was proposed to maintain the hydrophilicity of polycaprolactone (PCL) fibers, promoting cellular adhesion. Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs), cultured on aligned fibers, exhibited an elongated morphology with extension along the fiber axis. In comparison to Petri dishes and suspended random fiber scaffolds, hiPSC-CMs on suspended aligned fiber scaffolds demonstrated enhanced sarcomere organization, spontaneous synchronous contraction, and gene expression indicative of maturation. This work demonstrates the suspended and aligned nano-fibrous scaffold provides a more realistic biomimetic environment for hiPSC-CMs, which promoted further research on the inducing effect of fiber scaffolds on hiPSC-CMs microstructure and gene-level expression.
... In Yang et al., 2014, characteristics of adult CMs and immature CMs derived from hESCs and iPSCs were thoroughly and concisely summarized; we invite the readers here to refer to this previous review for details on, including metrics of, key maturation differences between hESC-and iPSC-CMs and adult CMs, as many of these specifics are beyond the scope of this review [8]. Another recent review, Sun and Nunes, 2017, also provided detailed comparisons of hESC-and iPSC-CMs to adult CMs in terms of maturation [37]. The comparison in Yang et al. included differences in CM size, shape, sarcomere structures, electrophysiological properties, binucleation, and abundance and distribution of mitochondria, as well as several cardiac genes that are upregulated in the adult heart compared to hESC-and iPSC-CMs, related to sarcomere and ion transporter functions [8]. ...
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Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be differentiated into cardiomyocytes (hESC-CMs and iPSC-CMs, respectively), which hold great promise for cardiac regenerative medicine and disease modeling efforts. However, the most widely employed differentiation protocols require undefined substrates that are derived from xenogeneic (animal) products, contaminating resultant hESC- and iPSC-CM cultures with xenogeneic proteins and limiting their clinical applicability. Additionally, typical hESC- and iPSC-CM protocols produce CMs that are significantly contaminated by non-CMs and that are immature, requiring lengthy maturation procedures. In this review, we will summarize recent studies that have investigated the ability of purified extracellular matrix (ECM) proteins to support hESC- and iPSC-CM differentiation, with a focus on commercially available ECM proteins and coatings to make such protocols widely available to researchers. The most promising of the substrates reviewed here include laminin-521 with laminin-221 together or Synthemax (a synthetic vitronectin-based peptide coating), which both resulted in highly pure CM cultures. Future efforts are needed to determine whether combinations of specific purified ECM proteins or derived peptides could further improve CM maturation and culture times, and significantly improve hESC- and iPSC-CM differentiation protocols.
... Regarding cardiac development, multiple factors including biophysical, biochemical or biological cues may influence cardiomyocyte maturation. Accordingly, previous studies have involved molecular targets, genetic manipulation methods, and cell co-culture or implantation to induce further maturation of iPSC-CMs, especially in structural or functional properties (Abilez et al., 2017;Sun and Nunes, 2017). However, these strategies are difficult to better recapitulate the in vivo myocardium environment. ...
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Although pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have been proved to be a new platform for heart regeneration, the lack of maturity significantly hinders the clinic application. Recent researches indicate that the function of stem cell is associated with the nanoscale geometry/topography of the extracellular matrix (ECM). However, the effects of 3D nanofibrous scaffolds in maturation of iPSC-CMs still remain unclear. Thus, we explored the effects of restructuring iPSC-CMs in 3D nano-scaffolds on cell morphology, cardiac-specific structural protein, gap junction and calcium transient kinetics. Using the electrospinning technology, poly-(ε-caprolactone) (PCL) nanofibrous scaffold were constructed and iPSC-CMs were seeded into these forms. As expected, strong sarcolemmal remodeling processes and myofilament reorientation were observed in 3D nano-scaffolds culture, as well as more expression of cardiac mature proteins, such as β-MHC and MLC2v. The mature morphology of 3D-shaped iPSC-CMs leaded to enhanced calcium transient kinetics, with increased calcium peak transient amplitude and the maximum upstroke velocity (Vmax). The results revealed that the maturation of iPSC-CMs was enhanced by the electrospun 3D PCL nanofibrous scaffolds treatment. These findings also proposed a feasible strategy to improve the myocardium bioengineering by combining stem cells with scaffolds.
... In contrast to the needle injection technique, a cell sheet can drive a large number of cells to damaged tissue without transplanted cell loss or injury to the host myocardium. Furthermore, the arrangement of hiPSC-CMs in three-dimensional patches promotes their continuous maturation [174,198,199]. ...
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Myocardial infarction is the main driver of heart failure due to ischemia and subsequent cell death, and cell-based strategies have emerged as promising therapeutic methods to replace dead tissue in cardiovascular diseases. Research in this field has been dramatically advanced by the development of laboratory-induced pluripotent stem cells (iPSCs) that harbor the capability to become any cell type. Like other experimental strategies, stem cell therapy must meet multiple requirements before reaching the clinical trial phase, and in vivo models are indispensable for ensuring the safety of such novel therapies. Specifically, translational studies in large animal models are necessary to fully evaluate the therapeutic potential of this approach; to empirically determine the optimal combination of cell types, supplementary factors, and delivery methods to maximize efficacy; and to stringently assess safety. In the present review, we summarize the main strategies employed to generate iPSCs and differentiate them into cardiomyocytes in large animal species; the most critical differences between using small versus large animal models for cardiovascular studies; and the strategies that have been pursued regarding implanted cells’ stage of differentiation, origin, and technical application.
... Maturation of hPSC-CM involves physiological hypertrophy associated with organization of sarcomeric structure, along with presence of T-tubules (71). hPSC-CM maturation also involves more efficient calcium handling, improved electrophysiological properties and higher contractile force (72). Therefore, transplanted CM with properties that more closely resemble adult myocardium would reduce the risk of arrhythmias and have improved contractile properties (73). ...
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Cardiovascular disease is the leading cause of death worldwide and bears an immense economic burden. Late-stage heart failure often requires total heart transplantation; however, due to donor shortages and lifelong immunosuppression, alternative cardiac regenerative therapies are in high demand. Human pluripotent stem cells (hPSCs), including human embryonic and induced pluripotent stem cells, have emerged as a viable source of human cardiomyocytes for transplantation. Recent developments in several mammalian models of cardiac injury have provided strong evidence of the therapeutic potential of hPSC-derived cardiomyocytes (hPSC-CM), showing their ability to electromechanically integrate with host cardiac tissue and promote functional recovery. In this review, we will discuss recent developments in hPSC-CM differentiation and transplantation strategies for delivery to the heart. We will highlight the mechanisms through which hPSC-CMs contribute to heart repair, review major challenges in successful transplantation of hPSC-CMs, and present solutions that are being explored to address these limitations. We end with a discussion of the clinical use of hPSC-CMs, including hurdles to clinical translation, current clinical trials, and future perspectives on hPSC-CM transplantation.
... For instance, we and others have reported that retinoic acid and fatty acid treatment could enhance the structural, metabolic and electrophysiological maturation of hPSC-CMs (Yang et al., 2019;Miao et al., 2020). In addition, tissue engineering, 3D culture and miRNAs were also used to promote hPSC-CMs maturation Sun and Nunes, 2017). However, much simpler and more effective approaches are urgently needed to drive hPSC-CM maturation at an affordable time and cost. ...
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Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) represent an infinite cell source for cardiovascular disease modeling, drug screening and cell therapy. Despite extensive efforts, current approaches have failed to generate hPSC-CMs with fully adult-like phenotypes in vitro, and the immature properties of hPSC-CMs in structure, metabolism and electrophysiology have long been impeding their basic and clinical applications. The prenatal-to-postnatal transition, accompanied by severe nutrient starvation and autophagosome formation in the heart, is believed to be a critical window for cardiomyocyte maturation. In this study, we developed a new strategy, mimicking the in vivo starvation event by Earle’s balanced salt solution (EBSS) treatment, to promote hPSC-CM maturation in vitro. We found that EBSS-induced starvation obviously activated autophagy and mitophagy in human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Intermittent starvation, via 2-h EBSS treatment per day for 10 days, significantly promoted the structural, metabolic and electrophysiological maturation of hESC-CMs. Structurally, the EBSS-treated hESC-CMs showed a larger cell size, more organized contractile cytoskeleton, higher ratio of multinucleation, and significantly increased expression of structure makers of cardiomyocytes. Metabolically, EBSS-induced starvation increased the mitochondrial content in hESC-CMs and promoted their capability of oxidative phosphorylation. Functionally, EBSS-induced starvation strengthened electrophysiological maturation, as indicated by the increased action potential duration at 90% and 50% repolarization and the calcium handling capacity. In conclusion, our data indicate that EBSS intermittent starvation is a simple and efficient approach to promote hESC-CM maturation in structure, metabolism and electrophysiology at an affordable time and cost.
... engineered heart tissue with higher force contraction. Sun and Nunes reviewed the different techniques to stimulate cells mechanically and electrically [91]. ...
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The pathophysiology of dilated cardiomyopathy (DCM), one major cause of heart failure, is characterized by the dilation of the heart but remains poorly understood because of the lack of adequate in vitro models. Current 2D models do not allow for the 3D organotypic organization of cardiomyocytes and do not reproduce the ECM perturbations. In this review, the different strategies to mimic the chemical, physical and topographical properties of the cardiac tissue affected by DCM are presented. The advantages and drawbacks of techniques generating anisotropy required for the cardiomyocytes alignment are discussed. In addition, the different methods creating macroporosity and favoring organotypic organization are compared. Besides, the advances in the induced pluripotent stem cells technology to generate cardiac cells from healthy or DCM patients will be described. Thanks to the biomaterial design, some features of the DCM extracellular matrix such as stiffness, porosity, topography or chemical changes can impact the cardiomyocytes function in vitro and increase their maturation. By mimicking the affected heart, both at the cellular and at the tissue level, 3D models will enable a better understanding of the pathology and favor the discovery of novel therapies.
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Backgrounds: -Tissue engineering enables the generation of functional human cardiac tissue using cells derived in vitro in combination with biocompatible materials. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes provide a cell source for cardiac tissue engineering; however, their immaturity limits their potential applications. Here we sought to study the effect of mechanical conditioning and electrical pacing on the maturation of hiPSC-derived cardiac tissues. METHODSS: -Cardiomyocytes derived from hiPSCs were used to generate collagen-based bioengineered human cardiac tissue. Engineered tissue constructs were subjected to different mechanical stress and electrical pacing conditions. RESULTSS: -The engineered human myocardium exhibits Frank-Starling-type force-length relationships. After 2 weeks of static stress conditioning, the engineered myocardium demonstrated increases in contractility (0.63±0.10 mN/mm(2) vs 0.055±0.009 mN/mm(2) for no stress), tensile stiffness, construct alignment, and cell size. Stress conditioning also increased SERCA2 expression, which correlated with a less negative force-frequency relationship. When electrical pacing was combined with static stress conditioning, the tissues showed an additional increase in force production (1.34±0.19 mN/mm(2)), with no change in construct alignment or cell size, suggesting maturation of excitation-contraction coupling. Supporting this notion, we found expression of RYR2 and SERCA2 further increased by combined static stress and electrical stimulation. Conclusions: -These studies demonstrate that electrical pacing and mechanical stimulation promote maturation of the structural, mechanical and force generation properties of hiPSC-derived cardiac tissues.
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The therapeutic success of human stem cell-derived cardiomyocytes critically depends on their ability to respond to and integrate with the surrounding electromechanical environment. Currently, the immaturity of human cardiomyocytes derived from stem cells limits their utility for regenerative medicine and biological research. We hypothesize that biomimetic electrical signals regulate the intrinsic beating properties of cardiomyocytes. Here we show that electrical conditioning of human stem cell-derived cardiomyocytes in three-dimensional culture promotes cardiomyocyte maturation, alters their automaticity and enhances connexin expression. Cardiomyocytes adapt their autonomous beating rate to the frequency at which they were stimulated, an effect mediated by the emergence of a rapidly depolarizing cell population, and the expression of hERG. This rate-adaptive behaviour is long lasting and transferable to the surrounding cardiomyocytes. Thus, electrical conditioning may be used to promote cardiomyocyte maturation and establish their automaticity, with implications for cell-based reduction of arrhythmia during heart regeneration.
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Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are the most promising source of cardiomyocytes (CMs) for experimental and clinical applications, but their use is largely limited by a structurally and functionally immature phenotype that most closely resembles embryonic or fetal heart cells. The application of physical stimuli to influence hPSC-CMs through mechanical and bioelectrical transduction offers a powerful strategy for promoting more developmentally mature CMs. Here we summarize the major events associated with in vivo heart maturation and structural development. We then review the developmental state of in vitro derived hPSC-CMs, while focusing on physical (electrical and mechanical) stimuli and contributory (metabolic and hypertrophic) factors that are actively involved in structural and functional adaptations of hPSC-CMs. Finally, we highlight areas for possible future investigation that should provide a better understanding of how physical stimuli may promote in vitro development and lead to mechanistic insights. Advances in the use of physical stimuli to promote developmental maturation will be required to overcome current limitations and significantly advance research of hPSC-CMs for cardiac disease modeling, in vitro drug screening, cardiotoxicity analysis and therapeutic applications.
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Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n = 96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n = 19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16–18 days at 0.5 Hz developed 2.2 × higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca2 +-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2 Hz in the first week and 1.5 Hz thereafter developed 1.5 × higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT.
The generation of human cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) has become an important resource for modeling human cardiac disease, and for drug screening and also holds significant potential for cardiac regeneration. Many challenges remain to be overcome however, before innovation in this field can translate into a change in the morbidity and mortality associated with heart disease. Of particular importance for the future application of this technology is an improved understanding of the electrophysiologic characteristics of CMs, so that better protocols can be developed and optimized for generating hPSC-CMs. Many different cell culture protocols are currently utilized to generate CMs from hPSCs and all appear to yield relatively “developmentally” immature CMs with highly heterogeneous electrical properties. These hPSC-CMs are characterized by spontaneous beating at highly variable rates with a broad range of depolarization–repolarization patterns suggestive of mixed populations containing atrial, ventricular and nodal cells. Many recent studies have attempted to introduce approaches to promote maturation and to create cells with specific functional properties. In this review, we summarize the studies in which the electrical properties of CMs derived from stem cells have been examined. In order to place this information in a useful context, we also review the electrical properties of CMs as they transition from the developing embryo to the adult human heart. The signal pathways involved in the regulation of ion channel expression during development are also briefly reviewed.
Human pluripotent stem cells (hPSCs)-derived cardiomyocytes (hPSC-CMs) represent a potential indefinite cell supply for cardiac tissue engineering and possibly regenerative medicine applications. However, these cells are immature compared with adult ventricular cardiomyocytes. In order to overcome this limitation, an engineered platform, called biowire, was devised to provide cultured cardiomyocytes important biomimetic cues present during embryo development, such as three-dimensional cell culture, extracellular matrix composition, soluble factors and pacing through electrical stimulation, to induce the maturation of hPSC-CMs in vitro.
Objective: The purpose of this study was to investigate the properties of the slow component of the delayed rectifier potassium current (IKs) in myocytes isolated from undiseased human left ventricles. Methods: The whole-cell configuration of the patch-clamp technique was applied in 58 left ventricular myocytes from 15 hearts at 37°C. Nisoldipine (1 μM) was used to block inward calcium current (ICa) and E-4031 (1–5 μM) was applied to inhibit the rapid component of the delayed rectifier potassium current (IKr). Results: In 31 myocytes, an E-4031 insensitive, but L-735,821 and chromanol 293B sensitive, tail current was identified which was attributed to the slow component of IK (IKs). Activation of IKs was slow (τ = 903±101 ms at 50 mV, n = 14), but deactivation of the current was relatively rapid (τ = 122.4±11.7 ms at −40 mV, n = 19). The activation of IKs was voltage independent but its deactivation showed clear voltage dependence. The deactivation was faster at negative voltages (about 100 ms at −50 mV) and slower at depolarized potentials (about 300 ms at 0 mV). In six cells, the reversal potential was −81.6±2.8 mV on an average which is close to the K+ equilibrium potential suggesting K+ as the main charge carrier. Conclusion: In undiseased human ventricular myocytes, IKs exhibits slow activation and fast deactivation kinetics. Therefore, in humans IKs differs from that reported in guinea pig, and it best resembles IKs described in dog and rabbit ventricular myocytes.