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Am J Physiol Heart Circ Physiol 303: H133–H143, 2012.
First published May 11, 2012; doi:10.1152/ajpheart.00007.2012.
Review
Physiological aspects of cardiac tissue engineering
Thomas Eschenhagen, Alexandra Eder, Ingra Vollert, and Arne Hansen
Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center Hamburg, University Medical
Center Hamburg Eppendorf, Hamburg, Germany
Submitted 4 January 2012; accepted in final form 17 April 2012
embryonic stem cells; induced pluripotent stem cells; heart; disease modeling; force
of contraction
THIS ARTICLE is part of a collection on Physiological Basis of
Cardiovascular Cell and Gene Therapies. Other articles appearing in this collection, as well as a full archive of all collections, can be found online at http://ajpheart.physiology.org/.
Introduction
Tissue engineering holds two promises: to generate surrogate three-dimensional (3-D) tissues for organ repair and experimental test beds modeling physiological organ function
better than standard two-dimensional (2-D) monolayer cell
cultures. A quarter century after coining the term “tissue
engineering,” initial overoptimistic expectations have made
way to a more realistic appraisal of its chances and limitations
(47). Here we review the current state of myocardial tissue
engineering for cardiac repair, preclinical drug development,
and disease modeling.
Tissue Engineering for Cardiac Repair: Different Approaches
Heart muscle cells lose their capacity to divide early after
birth (85), which makes their loss due to myocardial infarction,
increased hemodynamic load, infection, genetic disorders, or
toxins essentially irreversible. Whether the loss of myocytes is
indeed complete or residual regeneration is retained is an area
of active debate (3, 7, 38, 51, 59, 85), but there is no doubt that
loss of a relevant fraction of myocytes (e.g., during myocardial
Address for reprint requests and other correspondence: T. Eschenhagen,
Dept. of Experimental Pharmacology and Toxicology, Univ. Centre Hamburg
Eppendorf, Martinistr. 52, 20246 Hamburg, Germany (e-mail: t.eschenhagen
@uke.de).
http://www.ajpheart.org
infarction) leads to a permanent reduction in contractile function and eventually heart failure, a growing medical problem
worldwide. It is intuitively appealing, therefore, to substitute
the loss of myocytes by exogenous or endogenous sources, the
promise of “regenerative cardiology” (18, 63, 75, 86). In this
scenario, engineering techniques could play different roles.
The simplest and potentially most straightforward engineering technique is to create conditions in the (patient)
heart that promote cardiac myocyte progenitor cell homing
to the (injured) myocardium (in situ tissue engineering).
Examples are implantation of nanofibers (15), alginate (48),
or cardiac extracellular matrix scaffolds (29). The attraction
of this approach is that it is simple, can easily be done under
good manufacturing practice and good clinical practice
conditions, and can be marketed. Indeed, the first clinical
studies with alginate injections after myocardial infarction
are underway in Europe and Israel (clinicaltrials.gov,
NCT01226563, NCT01311791). The big question, however,
is whether cardiac progenitors exist in the adult heart at all
(particularly in elderly patients) and, if yes, whether their
homing and tissue building capacity can be promoted by
injecting extracellular matrixes or fibers. If not, the approach could still promote angiogenesis and/or homing of
nonmyocytes that, via paracrine factors, improve the survival of surrounding myocytes. None of these questions has
been unambiguously answered, but a recent publication
using an elegant genetic approach concluded that injected
hematopoietic cells do not transdifferentiate into myocytes
as initially suggested (63) but stimulate an (unidentified)
endogenous progenitor population to form new myocardium
after experimentally induced infarction in mice (51).
0363-6135/12 Copyright © 2012 the American Physiological Society
H133
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Eschenhagen T, Eder A, Vollert I, Hansen A. Physiological aspects of cardiac
tissue engineering. Am J Physiol Heart Circ Physiol 303: H133–H143, 2012. First
published May 11, 2012; doi:10.1152/ajpheart.00007.2012.—Cardiac tissue engineering aims at repairing the diseased heart and developing cardiac tissues for basic
research and predictive toxicology applications. Since the first description of
engineered heart tissue 15 years ago, major development steps were directed toward
these three goals. Technical innovations led to improved three-dimensional cardiac
tissue structure and near physiological contractile force development. Automation
and standardization allow medium throughput screening. Larger constructs composed of many small engineered heart tissues or stacked cell sheet tissues were
tested for cardiac repair and were associated with functional improvements in rats.
Whether these approaches can be simply transferred to larger animals or the human
patients remains to be tested. The availability of an unrestricted human cardiac
myocyte cell source from human embryonic stem cells or human-induced pluripotent stem cells is a major breakthrough. This review summarizes current tissue
engineering techniques with their strengths and limitations and possible future
applications.
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PHYSIOLOGICAL ASPECTS OF CARDIAC TISSUE ENGINEERING
size, and modulus of the construct. To date, however, the
ability to imitate the natural extracellular environment and the
microstructure of the heart remains imperfect at best. Moreover, a stereotypic observation with various solid matrixes is
that cells do not easily enter the matrix and distribute in free
spaces. Instead, they essentially seed on the surface and a few
micrometers beyond (49, 65, 72). Related to this issue is that
prefabricated porous matrixes prevent rather than promote
cell-cell contact by separating cells via matrix septa. Coupling
of cells is an essential requirement for tissue formation, and its
prevention by matrixes thus remains an unresolved conceptual
problem. A second matrix-based tissue engineering approach
replaces technical engineering of a nature-like matrix by decellularizing intact heart tissues, e.g., from pig hearts (64). A
relatively simple sodium dodecyl sulphate- and Triton X-100based procedure allows complete removal of the heart’s cellular components while preserving a surprisingly intact natural
matrix of both the myocardium and great blood vessels (64).
Whereas this approach elegantly solves the question of how to
obtain a nature-like matrix, it does not overcome the inherent
problems of having cells quantitatively enter the matrix. A
possible route could be provided by the intact blood vessels,
but here the advantage of preservation apparently turns into a
disadvantage, because cells when perfused into the (decellularized) coronary arteries did not quantitatively egress from the
circulation and invade the surrounding myocardial matrix (64).
Thus the dream of the complete, vascularized artificial heart
will likely remain unfulfilled for a while, but it will be interesting to see how this issue can be solved. Third, a conceptionally different matrix-based approach employs liquid hydrogels such as collagen I (8, 22, 110), matrigel (55), or fibrin to
make tissues ex vivo (34). Here freshly isolated cardiac cells
are mixed with natural, gel-forming, extracellular matrixes that
fulfill the main purpose of keeping cells in a 3-D space until
they form cell-cell contacts and generate their own extracellular matrix, thereby forming a tissue. The hydrogels also appear
to protect isolated cells from a process termed anoikis, cell
death due to loss of cell-cell contacts (41, 57). In the various
hydrogel approaches currently pursued, the macroscopic form
of the construct is defined by respective casting molds and the
techniques to fix the developing tissue to holders (Fig. 1). The
latter is crucial because good cardiac tissue development occurs only in constructs growing under continuous mechanical
load (8, 22, 26, 109), an observation also made with engineered
skeletal muscles (96 –97). Strain on the developing tissue
derives from cells forming contacts to neighboring cells and
remodeling the extracellular matrix, which leads to retraction
of the hydrogel and tension development if the ends are fixed
to something. In the absence of mechanical strain, cells degenerate and do not form a beating tissue (own unpublished
observation); in its presence a near physiological heart tissue
development occurs (Fig. 2). The heart consists of fibroblasts,
endothelial cells, and cardiomyocytes, raising the question as to
whether a certain ratio of these three cell populations supports
constitution of cardiac tissues in vitro. A few reports have addressed this question systematically and most support the hypothesis that endothelial cells and fibroblasts support tissue development (12, 60, 71, 94), but one also reported inferior outcomes in
the presence of fibroblasts (50), indicating that more work is
required to answer this question.
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The second engineering approach aims at increasing the
efficiency of (stem) cell application to the injured heart. Simple
cell injection into the beating myocardium leads to massive
rates of immediate cell death and very low retention rates over
time. Carefully performed studies report that values of ⬍10%
of cells remain a few days after cell injection in the heart of
animals (19, 69, 101) or intracoronary infusion in patients (36).
When using potentially tumorigenic cells such as embryonic
stem cells, the high washout rate not only reduces their potential therapeutic efficiency but also leads to cell deposition in the
lung, liver, kidney, and spleen (19) and constitutes a clear risk
of systemic tumor formation as demonstrated in mice (11).
Tissue engineering methods can improve cell retention rates.
Probably the most advanced approach in this respect is the cell
sheet technique (62, 83). Key to this technique was the development of thermosensitive culture dish surfaces that allow
detachment of intact cellular monolayers (of principally unlimited size in 2-D) at room temperature. The resulting thin sheets
can be directly attached to the beating heart without glue or
sutures. The group has developed semiautomated devices to
facilitate and standardize this procedure for clinical application
and reported the first implantation of cell sheets in a patient
with severe dilated cardiomyopathy (78). Despite the use of
autologous skeletal myoblasts that lack the capacity to differentiate into cardiac myocytes and do not couple to the host
myocardium (74), improvements of cardiac function were
reported. This and former studies with mesenchymal stroma
cells (53) or adipocytes (40) support the idea that the effects of
the cell sheet technique are, at least to a large part, due to
paracrine activity and mechanical stabilization of the scarred
ventricle (33). Advantages of the cell sheet technique are its
simplicity, the selection against dead cells in the preimplantation step in vitro, and very high cellular retention rate. Cell
sheets can also be stacked to form thicker tissues. Unfortunately, however, this possibility is, in the case of neonatal rat
heart cells, limited to 3 to 4 sheets (30 – 40 ␮m total thickness)
by the natural diffusion limits of factors necessary for and
released by metabolically active myocytes. A multistep surgical procedure in which cell sheets were stacked on each other
repeatedly with intervals sufficient to allow vascularization
overcame the limitation (82), but it is unlikely that a multistep
open-heart surgery could be transferred to patients. Alternative
ideas to increase cell retention in a cell therapy approach
include injection of cells in collagen, matrigel, fibrin, or other
extracellular matrices that form hydrogels (1, 14, 46). Whereas
the hydrogel certainly helps to retain cells in the area of
injection, it is less clear whether (and if yes, how) it can
overcome the high death rates induced by the pressure needed
to inject cells in a relatively stiff environment such as the
beating ventricular myocardium.
The third “traditional” tissue engineering technique aims at
building functional 3-D artificial heart tissues that can be
implanted onto the injured heart. Apparently, this is the most
ambitious and difficult goal and has been approached from
three different angles. One is to try to fabricate matrixes that
imitate the natural extracellular environment as closely as
possible so that cells can integrate into it and form a tissue.
Examples of prefabricated, solid matrixes are alginate (49),
polylactic acid (65), polyglycolic acid (66), and mixtures
thereof or collagen sponges (72). The great advantage of this
approach is the principally unlimited possibility to vary shape,
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PHYSIOLOGICAL ASPECTS OF CARDIAC TISSUE ENGINEERING
H135
Cardiac Repair with Engineered Tissues: Results from
Animal Experiments
Numerous studies tested engineered tissues or related products in small series of standard laboratory animals. Moreover,
composite cell sheets made from adipocytes and embryonic
stem cell-derived cardiovascular progenitors were implanted in
five infarcted nonhuman primates (6), and recently, the first
patient was treated with an autologous myoblast cell sheet (78).
All studies reported some functional benefit. However, studies
are lacking that compared the functional consequences of the
different cardiac tissue engineering approaches described
above systematically and in a statistically meaningful way.
Noncardiac cells (adipocytes) were applied as sheets or suspended cell injections in a direct comparison, showing better
results with the sheet technique (33). However, here the mechanism can only be paracrine or mechanic, a precluding con-
clusion on cardiac constructs. Thus, at this point, it is impossible to substantiate the widely held believe that tissue engineering provides a better means to repair a heart than direct
application of cells or that one technique is superior than
another. Yet, the published studies provide a number of important messages.
First, despite the lack of immediate vascularization (see
below), thin cell sheets (28, 82) and collagen I/matrigel-based
engineered heart tissue (EHT) (106, 109) survived when implanted in immunodeficient/-suppressed rats/mice (both on the
heart and extracardiac places) and formed new, relatively
well-organized heart tissue. The quality and thickness of the
new tissue was much better than anything published to date
with injected cells. EHTs from neonatal rat hearts induce
strong immune responses that need to be suppressed to preserve the tissue after implantation (21, 106). Interestingly,
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Fig. 1. Schematic illustration of different hydrogel-based cardiac tissue engineering approaches, cell sources, and techniques for
evaluation of contractile function. The principle is based on mixing isolated cells with
liquid hydrogel, which solidifies over time
(fast with fibrin, slower with collagen I). The
form of the final construct is determined by
the respective casting mold. Alignment of
cells and improved tissue quality are obtained
by subjecting the developing tissues to various form of mechanical load plus/minus electrical pacing. Cardiac function is either determined by manually attaching the tissues to
mechanical force transducers (A–C) or by
video-optical recordings (D and E). Force
calculation in the latter case takes into account the elastic properties of the posts to
which the growing tissues are attached and
against which they beat. NRCM, neonatal rat
cardiac myocytes; NMCM, neonatal mouse
cardiac myocytes; mESC, mouse embryonic
stem cells; hESC, human embryonic stem
cells; CM, cardiac myocytes; MSC, mesenchymal stem cells.
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PHYSIOLOGICAL ASPECTS OF CARDIAC TISSUE ENGINEERING
EHTs made from unfractionated neonatal rat hearts develop
vascular structures in vitro that appear to participate in the
formation of blood-perfused vasculature after implantation
onto the left ventricle (60, 81, 109). Similar findings were
made in stacked cell sheets from neonatal rat hearts (81) and
EHTs from human embryonic stem cell (hESC)-derived cardiac myocytes that were mixed with endothelial cells (12, 94).
Vascularization was extensive a few weeks after implantation
onto the heart (21, 81, 106, 109). The exact time course
remains insufficiently studied.
Second, implanted engineered tissues electrically connect to
the host heart (28, 109). This may seem trivial, but it is all but
that. On the one hand, the tissues are implanted on the pericardial surface of the heart and, in case of EHTs, contain a
continuous layer of epithelial-like cells on the outer surface.
Thus at least two nonmyocyte layers separate host and donor
myocytes from each other at the time of implantation. And
these layers appear to increase in thickness rather than disappear over time, because an apparently isolating fibrous layer
was consistently observed between the implant and the host
myocardium a few weeks after implantation. So how does
coupling occur? Extensive direct myocyte-to-myocyte coupling has never been observed by histological means, but some
“bridge-like” connections have been reported (106). Whether
they can account for the almost undelayed electrical conductance between host and donor tissue (28, 109) is unclear. An
alternative could be “electrotonic coupling” between host and
donor myocardium, which occurs within ⬃1 length constant
(0.15 mm to 0.3 mm) over the entire surface (23).
Third, implantation of multiloop EHTs (by fusion of five
ring-format EHTs) improved diastolic and systolic function in
infarcted rodent hearts (109). The effects of implanting large
multi-loop EHT on cardiac function were substantial (increase
in fractional area shortening by 30%) and included re-establishment of wall thickening of the scar at the implantation site
in systole, a sign of new vital myocardium. These magnetic
resonance imaging results were associated with the formation
of a layer of new myocardium at the implantation site (0.5–1
mm in histological sections), enough to explain the functional
improvement. Furthermore, control EHTs made from noncardiac myocytes or nonvital EHTs did not have the same effect.
The study can therefore serve as a proof of principle for the
therapeutic potential of myocardial tissue engineering. Yet,
others described similar functional consequences using noncardiac cell sheets (33, 40, 53, 80) without clear evidence for
the formation of new myocardium. Thus the mechanisms
which accounts for functional improvements after implanting
cardiac or noncardiac tissues onto the heart remain to be fully
deciphered.
Recent Improvements in Cardiac Tissue Engineering
For many years, myocardial tissue engineering was restricted to embryonic chicken and neonatal rat or mouse heart
cells, simply because a source for human cardiac myocytes was
unavailable. This has changed dramatically because of the
recent progress in stem cell biology. In 1998, the first hESCs
were described (92), and three years later cardiac myocytes
were generated from hESCs (44). hESCs provided, for the first
time, an unlimited cell source with an undisputed capacity to
differentiate into essentially all types of cells of the body
including cardiac myocytes. Their discovery boosted cardiac
tissue engineering by answering the question as to where the
several hundred millions of human cardiac myocytes that are
necessary to make large myocardial patches may come from
(107). The ethical concerns with hESC work stimulated the
search for pluripotent alternatives. These combined with the
technical experience acquired with mouse and hESC cultures
were important drivers of the seminal discovery of methods to
induce pluripotency in mouse and human somatic cells by
introducing a combination of pluripotency factors (89).
hESCs, at least in principle, solved the issue of a renewable
human cardiac myocyte source for cardiac regeneration and
human-induced pluripotent stem (hiPS) cells that of a patientderived autologous approach. Another important bottleneck
remained for years, the low efficiency of cardiac myocyte
differentiation from pluripotent stem cells. This problem was
solved by studies that carefully deciphered the factors that
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Fig. 2. Development of fibrin-based engineered heart tissues over time of cultivation (hematoxylin and eosin-stained longitudinal paraffin sections). Note the
increase in cell length, longitudinal orientation, and network formation paralleled by a reduction of extracellular space. The 80-day-old engineered heart tissue
exhibits near-physiological cell morphology with cross striation and dense tissue organization. Scale bar, 100 ␮m.
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PHYSIOLOGICAL ASPECTS OF CARDIAC TISSUE ENGINEERING
Open Questions on the Way to Clinical Application
1) Are tissue engineering approaches with actively contracting tissues really superior to simple cell injections/infusions,
transplantation of noncontracting tissues or standard medical
therapy? Head-to-head comparisons under well-controlled conditions are needed to answer these questions. Such studies will
require randomization, accurate and robust readouts, blinded
investigators, and large numbers of animals.
2) What is the optimal animal model to assess this question?
Differences in cardiac physiology and size make mice and rats
principally suboptimal models. Thus it seems unlikely that
human EHTs (which finally should be tested) can stably couple
to a heart that beats at 600 (mouse) or 300 (rat) beats/min,
respectively. A possibility is to transplant human EHT into
immunosuppressed primates, but for ethical and economic
reasons such experiments require careful exploitation of all
other options. For example, EHTs could be generated from
larger and clinically more relevant animals such as (neonatal)
rabbits, pigs, or dogs to be transplanted in an allogeneic
procedure. Nobody has yet produced tissues from these species
and own attempts with newborn pigs were unsuccessful (unpublished data). However, porcine iPS cells have been recently
established (24) and could be a good future source of allogeneic cardiac myocytes for clinically relevant preclinical studies
in pigs with heart failure.
3) Do recent growth factor-mediated cardiac differentiation
protocols completely eliminate pluripotent stem cells and
thereby the risk of teratoma formation of engineered myocardial tissues? Rigorous long-term experiments with a suitable
number of animals (immunodeficient mice, rats) are needed.
Recent antibody-based selection protocols may be helpful in
this respect (20).
4) How to drive hESC/hiPS cell-derived myocytes and
EHTs into advanced maturation? The 3-D environment per se,
mechanical loading and electrical stimulation have been identified as factors promoting maturation, but even in EHT or
similar constructs cardiac myocytes remain immature (79, 94).
Recent evidence suggests that neuregulin may improve ventricular specification (104), but more work is clearly needed in
this area.
5) Does the relatively high risk of chromosomal aberrations,
gene duplications, and mutations in iPS cell lines (30, 39)
affect the function of (terminally differentiated) iPS-derived
cardiac products and the outcome of implantation studies?
6) Are cardiac myocytes/tissues from autologous iPS cells
devoid of immune responses? Recent data suggest that this
does not need to be the case (102), but this may depend on the
type of differentiated cell. This is challenging in practice to
study because iPS cell lines would need to be generated from
animals that, several months later, would serve as their own
recipients.
7) An open question related to the previous is, Which of the
three pluripotent stem cell sources are best suited for clinical
applications? IPS cell products have the theoretical advantage
of being autologous. But besides this (still unproven) advantage, logistic factors rather argue against an autologous and for
an “off the shelf” approach. Specifically, the time needed to
generate iPS-derived cardiac tissues amounts to a minimum of
4 – 6 mo, potentially too long for patients suffering from a
severe myocardial infarction. Parthenogenetic stem cells (76)
have theoretical advantages in terms of antigenic heterogeneity, which would reduce the number needed for stem cell banks
(105).
8) Is it possible to produce large-enough tissues that have a
realistic chance to support the contractile function of the
injured human heart (wall thickness ⬎ 10 mm)? Present
engineered myocardial tissues do not exceed a thickness of
50 –200 ␮m in vitro because of the absence of perfusion and
biological diffusion limits (34, 70, 73, 82, 106). One possible
approach is to implant loose 3-D networks of relatively immature cells that, after connection to the host circulation will
hypertrophy, thereby increase tissue density, as observed in a
study with multiloop EHTs (109). An alternative is to develop
methods to integrate functional blood vessels into 3-D cardiac
constructs, perfuse them in vitro, and connect the “vascularized” cardiac tissue to the host circulation. Materials created
using 3-D printing techniques (61) and decellularized natural
vascular beds (29) are at hand. An elegant alternative in vivo
prevascularization approach has been described recently (55).
Neonatal rat heart cells were mixed with matrigel and poured
into a chamber implanted close to an atrioventricular loop that
had been surgically constructed from the epigastric artery and
vein (55). This led to the formation of a well-developed, thick
(up to 2 mm) cardiac tissue with an autologous vascular bed,
suitable for implantation and connection to the host circulation.
9) Can engineered cardiac tissue be induced to integrate
correctly into the complex architecture of the heart? In contrast
to skeletal muscles in which fibers are strictly longitudinally
oriented, heart muscle strands rather form flat bands that are
wrapped around a cavity and can be oriented perpendicular to
each other in different layers. Theoretically, such architecture
could be mimicked using a completely synthetic matrix-based
approach (e.g., 3-D printing) or decellularized hearts, but it
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guide the earliest steps of cardiac development under normal
conditions and translated this knowledge into cell culture
protocols [e.g., (56)]. Recent multistep protocols with growth
factors that sequentially drive mesodermal and cardiac specification increased cardiac myocyte differentiation rates from
⬃1% (44) to 50% or more (100). By optimization of growth
factor combinations and timing, cardiac myocyte differentiation rates can be increased even further and, importantly, the
new protocols can be applied to essentially all pluripotent stem
cell lines including hiPS (42).
The progress in stem cell technology was crucial for the
engineering of human heart muscle for several reasons. First,
the high purity of cardiac myocytes allowed human EHT to be
generated without further purification (79, 94). This has obviated the need for manual dissection of beating clusters (12) or
genetic selection (2) with their inherent limitations. It has also
improved tissue homogeneity compared with the early work
with hESC-derived cells (12–13, 87). Second, just as important, the higher cardiac myocyte yield has allowed routine
work with engineered human heart muscles, opening new in
vitro applications (see below). Third, perspectively, the high
efficiency of growth factor-based differentiation protocols shall
substantially reduce the risk of teratoma formation by driving
pluripotent cells quantitatively into the mesenchymal or cardiac lineage (see below). More work is needed to substantiate
this assumption.
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PHYSIOLOGICAL ASPECTS OF CARDIAC TISSUE ENGINEERING
remains unclear whether cells applied to such constructs would
automatically follow the scaffold orientation. Hydrogel-based
methods cannot mimic the complex heart architecture a priori
but, when supplying cells in the gel with a directed mechanical
load, induce a strict longitudinal tissue orientation (34, 110).
Whether this orientation will remain after implanting the tissues on the heart or adapt to new strain lines of the host
environment remains unknown.
10) Could a cardiac patch be fixed onto the endocardium
(which is the first tissue to be affected by ischemia) rather than
the epicardium? And would it yield better results despite the
clearly more invasive technique?
Whereas cardiac regeneration may be the more exciting
goal, modeling heart tissue function in the dish is probably a
more realistic and short-term application of engineered cardiac
tissues. Indeed, we believe that 3-D cardiac tissues will soon play
a role in preclinical drug development and iPS cell-mediated
disease modeling. The main promise of the in vitro application
was defined in the first tissue engineering conference in 1987
(http://www.nsf.gov/pubs/2004/nsf0450/emergence.pdf) and,
specifically for the cardiac field, 15 years ago (22). Engineered
3-D tissues should mimic the natural cell environment better
than 2-D cultures. Theoretical advantages include the following. First, cells in a 3-D tissue can communicate and make
contacts with surrounding cells in all directions. In contrast,
cells in standard monolayer cell cultures sit on a rigid plastic
surface and have only side-to-side contacts with neighboring
cells. Second, cells in 3-D form an organized structure (22) and
proliferate less, which allows for long-term studies and alleviates the need for pharmacological suppression of mitotic activity as frequently done in 2-D cultures. Third, the 3-D
environment may promote differentiation of cardiac myocytes
toward a fully mature phenotype relative to 2-D. Evidence
supporting this hypothesis exists (93), but studies with hESCor hiPS-derived myocytes also show that 3-D alone is not the
cue for terminal maturation (79, 94). Fourth, contractile function measurements can be made more accurately in 3-D than in
2-D cultures. The initial hope was to establish 3-D engineered
cardiac tissues as an experimental model placed between standard 2-D cultures and more complex experimental cardiac
systems such as Purkinje fibers, papillary muscles/trabeculae,
Langendorff-perfused hearts, or the entire animal. The following paragraphs summarize how much of the promise of 3-D
tissues as models for cardiac muscle function has been fulfilled. Almost all data were derived from experiments using the
hydrogel approach, which, in contrast to other tissue engineering techniques, has been designed for functional measurements
of 3-D cardiac tissues in vitro and has found widespread
application. Over the years, the original lattice approach (22)
was modified and developed toward an improved in vitro assay
by modifying cell source, casting molds, cellular composition,
mode of stretching, force measurement, etc. (Fig. 1). Condensation of the hydrogel and degradation of the exogenous
extracellular matrix are essential aspects of the formation of a
densely packed cardiac tissue in vitro (Fig. 2). The mechanisms
underlying this process are poorly understood and need to be
studied systematically.
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Tissue Engineering to Model (Human) Heart Tissue Function
Force development. The fundamental function of the heart is
the development of contractile force. Based on single cell
analysis human ventricular cardiomyocytes develop a force of
50 mN/mm2 under optimized preload (95). Intact human trabeculae in an organ bath usually develop between 1 and 10
mN, which, if very thin, well-oxygenated muscles are used,
amounts to similar relative values [e.g., 44 mN/mm2 (35)].
Tissue-engineered muscles, in contrast, generally develop
much smaller forces, ranging between 0.1 and 4 mN/mm2
(Table 1). One reason is that the fraction of the engineered
cardiac construct populated by cells and compact muscle
strands is generally much smaller than in normal hearts. The
rest is cell-free extracellular matrix, which does not contribute
to contractile force but to tissue diameter (denominator) and to
passive stiffness. In consequence, active/passive force ratios
are often smaller in large engineered tissues (Table 1) than
values reported from nonfailing, ischemic and dilated cardiomyopathy heart preparations, amounting to 1–2 (37, 98). Nearphysiological force values have only been measured in ultrathin constructs (5) or aged EHTs almost devoid of free extracellular matrix (34). Inhomogenous cell distribution and low
cellular density in engineered heart constructs are due to the
fact that cells in engineered 3-D tissues rely exclusively on
diffusion as the supply of nutrients and oxygen, which is only
sufficient within the most peripheral 50 –100 ␮m of a tissue or
a muscle strand inside a tissue. Thus thicker constructs, particularly those made from preformed matrices, show a steep
gradient of cell density from the surface to the core (22, 73, 87,
94). The conclusion from these findings is the contractile force
of engineered cardiac constructs can approach physiological
values if constructs are sufficiently small and extracellular
matrix content low but does not simply increase with the size
of the construct. Another reason for subnormal forces reported
in many studies is that, in the absence of stretch or other means
to direct cells, cells are isotropically oriented and thus do not
transmit their contractile force in a coordinate manner. Force
measurements in such constructs are therefore relatively meaningless. The immaturity of cardiac myocytes likely also accounts for lower force development. In normal adult cardiac
myocytes with their almost crystalline organization the myofilament:mitochondria:nucleus ratio is 47:36:2 with the remaining consisting of 3.5% sarcoplasmic reticulum and 11.5%
cytosol (43). In myocytes visualized in sections from rat EHTs
by transmission electron microscopy, the ratio was 45:24:9
with 23% for sarcoplasmic reticulum and cytosol (110). These
values have not been quantitatively determined in constructs
from hESCs or hiPS cells but are considerably lower, indicating the low degree of maturity.
Reaction to physiological interventions. Cardiomyocytes, similar to skeletal muscle cells, increase active force development in
response to increased preload (Frank-Starling mechanism). Several studies have shown the same to be true in engineered cardiac
constructs [Table 1 and (4, 26, 50, 94, 108, 110)] and reported
increases between 1.9 –10-fold. The substantial variation is likely
in part due to practical difficulties in defining baseline values for
force development. A second physiological response is the forcefrequency relationship, in which a normal human heart muscle
increases force with increasing stimulation rate (Bowditch phenomenon 1871, http://vlp.mpiwg-berlin.mpg.de/library/data/
lit1387/index_html?pn⫽1&ws⫽1.5). Engineered cardiac tissues
show the same response, albeit at different directions (Table 1).
Review
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PHYSIOLOGICAL ASPECTS OF CARDIAC TISSUE ENGINEERING
Table 1. Physiological and pharmacological features of engineered cardiac constructs
Parameter
Active force/diameter
Force (active/passive/ratio)
Species
Quantification
Zimmermann et al., 2002 (110)
Baar et al., 2005 (5)
Feinberg et al., 2007 (25)
Hansen et al., 2010 (34)
Boudou et al., 2012 (9)
Asnes et al., 2006 (4)
de Lange et al., 2011 (16)
Tulloch et al., 2011 (94)
Schaaf et al., 2011 (79)
Zimmermann et al., 2000 (108)
Zimmermann et al., 2002 (110)
Baar et al., 2005 (5)
Hansen et al., 2010 (34)
Eschenhagen et al., 1997 (22)
Asnes et al., 2006 (4)
de Lange et al., 2011 (16)
Tulloch et al., 2011 (94)
Rat
Rat
Rat
Rat
Rat
Chicken
Mouse
Human
Human
Rat
Rat
Rat
Rat
Chicken
Chicken
Mouse
Human
Zimmermann et al., 2000 (108)
Fink et al., 2000 (26)
Fink et al., 2000 (26)
Asnes et al., 2006 (4)
Liau et al., 2011 (50)
de Lange et al., 2011 (16)
Tulloch et al., 2011 (94)
Rat
Rat
Chicken
Chicken
Mouse
Mouse
Human
Fink et al., 2000 (26)
Zimmermann et al., 2002 (110)
Münzel et al., 2005 (58)
Baar et al., 2005 (5)
Naito et al., 2006 (60)
Fink et al., 2000 (26)
Rat
Rat
Rat
Rat
Rat
Chicken
Zimmermann et al., 2000 (108)
Fink et al., 2000 (26)
Baar et al., 2005 (5)
Zimmermann et al., 2006 (109)
Fink et al., 2000 (26)
Eschenhagen et al., 1997 (22)
Schaaf et al., 2011 (79)
de Lange et al., 2011 (16)
Zimmermann et al., 2000 (108)
Pong et al., 2011 (68)
Eschenhagen et al., 1997 (22)
de Lange et al., 2011 (16)
Liau et al., 2011 (50)
—
Rat
Rat
Rat
Rat
Chicken
Chicken
Human
Mouse
Rat
Rat
Chicken
Mouse
Mouse
—
2 mN/mm2
66.2 mN/ mm2
1–4 mN/mm2
28.7 mN/mm2
0.96 mN/mm2
0.91 mN/mm2
1.19 mN/mm2
0.08 mN/mm2
0.12 mN/mm2
0.46 mN/0.63 mN/0.73
0.75 mN/0.27 mN/2.5
0.14 mN/0.26 mN/0.54
0.9 mN/0.75 mN/1.2
0.2 mN/2.81 mN/0.07
1 mN/4.1 mN/0.24
1 mN/1.1 mN/0.9
0.08 mN/0.4 mN/0.2
Fold increase
2.1-fold
3.4-fold
2-fold
3-fold
1.9-fold
10-fold
8-fold
EC50, fold increase
ND, 1.19-fold
3 nM, 2.5 fold
2 nM, 2.1 fold
ND, 2.5 fold
3 nM, 1.5-fold
ND, 1.48-fold
EC50, fold increase
0.24 mM, 5-fold
0.4 mM, 1.8-fold
5 mM
0.4 mM, 1.5-fold
3.7 mM, 2.2-fold
4 mM, 3-fold
0.8 mM, 7-fold
No change
Negative
Negative
Positive
Negative
Negative
—
Frank-Starling Mechanism
Isoprenaline
Calcium
FDAR
Force-frequency relationship
Post-rest potentiation
FDAR, frequency-dependent acceleration of relaxation; ND, no data.
Whereas chicken EHTs show a positive staircase (between 0.8
and 2 Hz), EHTs from rat and mouse exhibit a negative correlation between 1 and 5 Hz. Myocytes also increase their contraction
kinetics with increasing frequency, a phenomenon called frequency-dependent acceleration of relaxation (17). One report in mouse
EHTs did not observe frequency-dependent acceleration of relaxation between 6 and 8 Hz (16), but systematic studies over larger
ranges of frequency are lacking.
Reaction to pharmacological interventions. Cardiac, in contrast to skeletal, muscle is exquisitely depending on extracellular Ca2⫹-concentrations. A number of tissue engineering
approaches analyzed the relationship between force development and changes in extracellular Ca2⫹ concentration (22, 26,
68, 79, 108 –110). These experiments demonstrate that engineered neonatal rat cardiac tissues respond to changes in
extracellular Ca2⫹ similar to intact cardiac tissues of the rat.
However, a frequent observation in these studies was a hypersensitivity to extracellular Ca2⫹, i.e., a leftward shift of the
force-Ca2⫹ relationship compared with adult rat heart preparations. One factor accounting for this observation may be the
developmental stage of engineered cardiac constructs. Whereas
one study in neonatal rat heart preparations did not show Ca2⫹
hypersensitivity (77), others observed a marked leftward shift
of the force-Ca2⫹ relationship in skinned myofibers from
embryonic (embryonic day 16.5) and fetal mouse (embryonic
day 19.5) hearts compared with neonatal (postnatal day 7) or,
even more pronounced, to adult mouse hearts [postnatal weeks
6 – 8 (84)]. These findings were associated with differences in
the expression pattern of sarcomeric proteins, indicating that
myofilament composition contributes to higher Ca2⫹ sensitivity in the immature heart. Additionally, the relative importance
of sarcoplasmic reticulum and sarcolemmal Ca2⫹ cycling dif-
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00007.2012 • www.ajpheart.org
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Study
Review
H140
PHYSIOLOGICAL ASPECTS OF CARDIAC TISSUE ENGINEERING
In summary, engineered cardiac constructs have gained
increasing importance as advanced experimental test beds and
as material for cardiac repair. Since their first description 15
years ago, four major developments toward applications have
occurred. First, addition of mechanical loading and electrical
stimulation, higher O2-content, various medium supplements
and/or miniaturization have led to improved 3-D cardiac tissue
structure, maturation of myocytes, reduced extracellular matrix
content, and development of near-physiological contractile
force. Second, automation and standardization helped to set up
an assay format suitable for medium throughput screening.
Third, larger constructs composed of many small EHTs or
stacked cell sheet tissues have been tested for cardiac repair
and are associated with functional improvements in rats.
Whether these approaches can be simply transferred to larger
animals or the human patients remains to be tested. Theoretical
arguments strongly suggest the necessity for preimplantation
vascularization. Fourth, an unrestricted human cardiac myocyte cell source from hESCs or hiPS cells is available and
represents a major breakthrough. Whereas drug testing with
human engineered cardiac constructs is already reality, cardiac
regeneration with such constructs remains a fascinating yet
open perspective.
GRANTS
This work is supported by grants from the European Union (Angioscaff and
Biodesign), the German Research Foundation (DFG Es 88/12-1, DFG Ha 3/1),
the German Ministry of Research and Education [Deutsches Zentrum für
Herz-Kreislauf-Forschung e.V. (German Centre for Cardiovascular Research)], and the German Heart Foundation.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
T.E. conception and design of research; T.E. and A.H. drafted manuscript;
T.E., A.E., I.V., and A.H. edited and revised manuscript; T.E., A.E., I.V., and
A.H. approved final version of manuscript; A.E. performed experiments; A.E.
and I.V. prepared figures.
REFERENCES
1. Anderl JN, Robey TE, Stayton PS, Murry CE. Retention and biodistribution of microspheres injected into ischemic myocardium. J Biomed
Mater Res A 88: 704 –710, 2009.
2. Anderson D, Self T, Mellor IR, Goh G, Hill SJ, Denning C. Transgenic enrichment of cardiomyocytes from human embryonic stem cells.
Mol Ther 15: 2027–2036, 2007.
3. Anversa P, Kajstura J. Ventricular myocytes are not terminally differentiated in the adult mammalian heart. Circ Res 83: 1–14, 1998.
4. Asnes CF, Marquez JP, Elson EL, Wakatsuki T. Reconstitution of the
Frank-Starling mechanism in engineered heart tissues. Biophys J 91:
1800 –1810, 2006.
5. Baar K, Birla R, Boluyt MO, Borschel GH, Arruda EM, Dennis RG.
Self-organization of rat cardiac cells into contractile 3-D cardiac tissue.
FASEB J 19: 275–277, 2005.
6. Bel A, Planat-Bernard V, Saito A, Bonnevie L, Bellamy V, Sabbah L,
Bellabas L, Brinon B, Vanneaux V, Pradeau P, Peyrard S, Larghero
J, Pouly J, Binder P, Garcia S, Shimizu T, Sawa Y, Okano T,
Bruneval P, Desnos M, Hagege AA, Casteilla L, Puceat M, Menasche
P. Composite cell sheets: a further step toward safe and effective
myocardial regeneration by cardiac progenitors derived from embryonic
stem cells. Circulation 122: S118 –S123, 2010.
7. Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabe-Heider
F, Walsh S, Zupicich J, Alkass K, Buchholz BA, Druid H, Jovinge S,
Frisen J. Evidence for cardiomyocyte renewal in humans. Science 324:
98 –102, 2009.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00007.2012 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 16, 2017
fers between embryonic/fetal and adult hearts with the sarcoplasmic reticulum becoming increasingly dominant with maturation (88). This may also affect the sensitivity of intact
preparations to external Ca2⫹. To which extent these factors
account for the unusual Ca2⫹ sensitivity of engineered cardiac
constructs has not been addressed so far.
Several reports describe the existence of a sympathetic and
parasympathetic receptor system on cardiac constructs (26, 60,
110) by demonstrating positive inotropic and chronotropic
effects of isoprenaline and reversion by carbachol. The calculated half-maximal effective concentration for isoprenaline in
these experiments was shown to be in the lower nanomolar
range, which is ⬃10-fold less than those reported from studies
in neonatal rat hearts and adult rat papillary muscle [⬃50 nM;
(52, 91)]. The relative isoprenaline-induced increase in force
appears smaller in engineered constructs than in adult heart
preparations (⬃2-fold vs. 4 –5-fold, Table 1), which may again
indicate immaturity. But it also relates to the increased sensitivity to external Ca2⫹. For example, isoprenaline has no
inotropic effect at all at physiological Ca2⫹ concentrations of
1.8 mM, because contractile force is already maximal and
cannot be further enhanced.
Action potential and ion currents. Action potentials of cardiomyocytes are driven by a unique set of Na⫹, Ca2⫹, and K⫹
channels. Cardiac ion channel equipment and action potential
characteristics differ considerably between laboratory animals
(particularly mice and rats) and humans, developmental stages,
cardiac regions, and disease states (90). A typical example is
the transient outward repolarizing K⫹ current, which is dominant in mice and rats and small in humans and guinea pig,
strong in subepicardial and small in subendocardial layers and
downregulated in failing human hearts (99). The advent of
cardiac tissue engineering from human pluripotent cells is
raising the question as to whether these cells/engineered tissues
are electrophysiologically similar to adult human cardiomyocytes to serve as a better test platform than current rodentbased models. In fact, this question is very difficult to answer.
First, cardiomyocytes derived from human pluripotent stem
cells are heterogenous with some showing a more ventriclelike, others atrial-like, Purkinje-like, or pacemaker-like phenotype (67). The terminology shows already that these cells are
neither/nor. Rather they are electrophysiologically immature
cells resembling cardiomyocytes of 16-wk-old embryos (56).
This immaturity is reflected by low maximal diastolic potentials because of insufficient inwardly rectifying K⫹ current and
slow upstroke velocities (79, 103–104). Evidence suggests that
prolonged culturing (27, 54), addition of nonmyocytes (45),
and the 3-D format in EHT promote some aspects of maturation. Zhu et al. (104) demonstrated that the differentiation in the
presence of neuregulin increased the percentage of mature cardiomyocytes with slower firing rate, increased upstroke velocities,
and more negative maximum diastolic potential. Continuous electrical pacing improved neonatal rat cardiac constructs by transcriptional, histological, and contractile means (72) and may be a
way to improve cardiomyocytes maturation in human constructs
as well. Despite the immaturity of human cardiac myocytes from
pluripotent stem cells, several studies showed effects of indicator
drugs on single cardiomyocytes or engineered tissues (10, 31–32,
79). This opens the realistic perspective for their use in drug
development and toxicology applications.
Review
PHYSIOLOGICAL ASPECTS OF CARDIAC TISSUE ENGINEERING
29. Godier-Furnemont AF, Martens TP, Koeckert MS, Wan L, Parks J,
Arai K, Zhang G, Hudson B, Homma S, Vunjak-Novakovic G.
Composite scaffold provides a cell delivery platform for cardiovascular
repair. Proc Natl Acad Sci USA 108: 7974 –7979, 2011.
30. Gore A, Li Z, Fung HL, Young JE, Agarwal S, Antosiewicz-Bourget
J, Canto I, Giorgetti A, Israel MA, Kiskinis E, Lee JH, Loh YH,
Manos PD, Montserrat N, Panopoulos AD, Ruiz S, Wilbert ML, Yu
J, Kirkness EF, Izpisua Belmonte JC, Rossi DJ, Thomson JA, Eggan
K, Daley GQ, Goldstein LS, Zhang K. Somatic coding mutations in
human induced pluripotent stem cells. Nature 471: 63–67, 2011.
31. Guo L, Abrams RM, Babiarz JE, Cohen JD, Kameoka S, Sanders
MJ, Chiao E, Kolaja KL. Estimating the risk of drug-induced proarrhythmia using human induced pluripotent stem cell-derived cardiomyocytes. Toxicol Sci 123: 281–289, 2011.
32. Guo XM, Zhao YS, Chang HX, Wang CY, ELL, Zhang XA, Duan
CM, Dong LZ, Jiang H, Li J, Song Y, Yang XJ. Creation of engineered
cardiac tissue in vitro from mouse embryonic stem cells. Circulation 113:
2229 –2237, 2006.
33. Hamdi H, Planat-Benard V, Bel A, Puymirat E, Geha R, Pidial L,
Nematalla H, Bellamy V, Bouaziz P, Peyrard S, Casteilla L, Bruneval
P, Hagege AA, Agbulut O, Menasche P. Epicardial adipose stem cell
sheets results in greater post-infarction survival than intramyocardial
injections. Cardiovasc Res 91: 483–491, 2011.
34. Hansen A, Eder A, Bonstrup M, Flato M, Mewe M, Schaaf S,
Aksehirlioglu B, Schwoerer AP, Uebeler J, Eschenhagen T. Development of a drug screening platform based on engineered heart tissue.
Circ Res 107: 35–44, 2010.
35. Hasenfuss G, Mulieri LA, Blanchard EM, Holubarsch C, Leavitt BJ,
Ittleman F, Alpert NR. Energetics of isometric force development in
control and volume-overload human myocardium. Comparison with
animal species. Circ Res 68: 836 –846, 1991.
36. Hofmann M, Wollert KC, Meyer GP, Menke A, Arseniev L, Hertenstein B, Ganser A, Knapp WH, Drexler H. Monitoring of bone marrow
cell homing into the infarcted human myocardium. Circulation 111:
2198 –2202, 2005.
37. Holubarsch C, Ruf T, Goldstein DJ, Ashton RC, Nickl W, Pieske B,
Pioch K, Ludemann J, Wiesner S, Hasenfuss G, Posival H, Just H,
Burkhoff D. Existence of the Frank-Starling mechanism in the failing
human heart. Investigations on the organ, tissue, and sarcomere levels.
Circulation 94: 683–689, 1996.
38. Hsieh PC, Segers VF, Davis ME, MacGillivray C, Gannon J, Molkentin JD, Robbins J, Lee RT. Evidence from a genetic fate-mapping
study that stem cells refresh adult mammalian cardiomyocytes after
injury. Nat Med 13: 970 –974, 2007.
39. Hussein SM, Batada NN, Vuoristo S, Ching RW, Autio R, Narva E,
Ng S, Sourour M, Hamalainen R, Olsson C, Lundin K, Mikkola M,
Trokovic R, Peitz M, Brustle O, Bazett-Jones DP, Alitalo K, Lahesmaa R, Nagy A, Otonkoski T. Copy number variation and selection
during reprogramming to pluripotency. Nature 471: 58 –62, 2011.
40. Imanishi Y, Miyagawa S, Maeda N, Fukushima S, KitagawaSakakida S, Daimon T, Hirata A, Shimizu T, Okano T, Shimomura
I, Sawa Y. Induced adipocyte cell-sheet ameliorates cardiac dysfunction
in a mouse myocardial infarction model: a novel drug delivery system for
heart failure. Circulation 124: S10 –S17, 2011.
41. Karoubi G, Ormiston ML, Stewart DJ, Courtman DW. Single-cell
hydrogel encapsulation for enhanced survival of human marrow stromal
cells. Biomaterials 30: 5445–5455, 2009.
42. Kattman SJ, Witty AD, Gagliardi M, Dubois NC, Niapour M, Hotta
A, Ellis J, Keller G. Stage-specific optimization of activin/nodal and
BMP signaling promotes cardiac differentiation of mouse and human
pluripotent stem cell lines. Cell Stem Cell 8: 228 –240, 2011.
43. Katz AM. Structure of the heart. In: Physiology of the Heart (2nd ed.).
New York: Raven, 1992.
44. Kehat I, Kenyagin-Karsenti D, Snir M, Segev H, Amit M, Gepstein
A, Livne E, Binah O, Itskovitz-Eldor J, Gepstein L. Human embryonic stem cells can differentiate into myocytes with structural and
functional properties of cardiomyocytes. J Clin Invest 108: 407–414,
2001.
45. Kim C, Majdi M, Xia P, Wei KA, Talantova M, Spiering S, Nelson
B, Mercola M, Chen HS. Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation. Stem Cells Dev 19: 783–795, 2010.
46. Kofidis T, Lebl DR, Martinez EC, Hoyt G, Tanaka M, Robbins RC.
Novel injectable bioartificial tissue facilitates targeted, less invasive,
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00007.2012 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 16, 2017
8. Bian W, Liau B, Badie N, Bursac N. Mesoscopic hydrogel molding to
control the 3D geometry of bioartificial muscle tissues. Nat Protoc 4:
1522–1534, 2009.
9. Boudou T, Legant WR, Mu A, Borochin MA, Thavandiran N,
Radisic M, Zandstra PW, Epstein JA, Margulies KB, Chen CS. A
microfabricated platform to measure and manipulate the mechanics of
engineered cardiac microtissues. Tissue Eng Part A 18: 910 –919, 2012.
10. Braam SR, Tertoolen L, van de Stolpe A, Meyer T, Passier R,
Mummery CL. Prediction of drug-induced cardiotoxicity using human
embryonic stem cell-derived cardiomyocytes. Stem Cell Res 4: 107–116,
2010.
11. Cao F, van der Bogt KE, Sadrzadeh A, Xie X, Sheikh AY, Wang H,
Connolly AJ, Robbins RC, Wu JC. Spatial and temporal kinetics of
teratoma formation from murine embryonic stem cell transplantation.
Stem Cells Dev 16: 883–891, 2007.
12. Caspi O, Lesman A, Basevitch Y, Gepstein A, Arbel G, Habib IH,
Gepstein L, Levenberg S. Tissue engineering of vascularized cardiac
muscle from human embryonic stem cells. Circ Res 100: 263–272, 2007.
13. Chen QZ, Ishii H, Thouas GA, Lyon AR, Wright JS, Blaker JJ,
Chrzanowski W, Boccaccini AR, Ali NN, Knowles JC, Harding SE.
An elastomeric patch derived from poly(glycerol sebacate) for delivery
of embryonic stem cells to the heart. Biomaterials 31: 3885–3893, 2010.
14. Danoviz ME, Nakamuta JS, Marques FL, dos Santos L, Alvarenga
EC, dos Santos AA, Antonio EL, Schettert IT, Tucci PJ, Krieger JE.
Rat adipose tissue-derived stem cells transplantation attenuates cardiac
dysfunction post infarction and biopolymers enhance cell retention. PLoS
One 5: e12077, 2010.
15. Davis ME, Hsieh PC, Grodzinsky AJ, Lee RT. Custom design of the
cardiac microenvironment with biomaterials. Circ Res 97: 8 –15, 2005.
16. de Lange WJ, Hegge LF, Grimes AC, Tong CW, Brost TM, Moss
RL, Ralphe JC. Neonatal mouse-derived engineered cardiac tissue: a
novel model system for studying genetic heart disease. Circ Res 109:
8 –19, 2011.
17. DeSantiago J, Maier LS, Bers DM. Frequency-dependent acceleration
of relaxation in the heart depends on CaMKII, but not phospholamban. J
Mol Cell Cardiol 34: 975–984, 2002.
18. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the
scientific foundations of cardiac repair. J Clin Invest 115: 572–583, 2005.
19. Dow J, Simkhovich BZ, Kedes L, Kloner RA. Washout of transplanted
cells from the heart: a potential new hurdle for cell transplantation
therapy. Cardiovasc Res 67: 301–307, 2005.
20. Dubois NC, Craft AM, Sharma P, Elliott DA, Stanley EG, Elefanty
AG, Gramolini A, Keller G. SIRPA is a specific cell-surface marker for
isolating cardiomyocytes derived from human pluripotent stem cells. Nat
Biotechnol 29: 1011–1018, 2011.
21. Eschenhagen T, Didie M, Munzel F, Schubert P, Schneiderbanger K,
Zimmermann WH. 3D engineered heart tissue for replacement therapy.
Basic Res Cardiol 97, Suppl 1: I146 –I152, 2002.
22. Eschenhagen T, Fink C, Remmers U, Scholz H, Wattchow J, Weil J,
Zimmermann W, Dohmen HH, Schafer H, Bishopric N, Wakatsuki
T, Elson EL. Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system. FASEB J
11: 683–694, 1997.
23. Eschenhagen T, Zimmermann WH, Kleber AG. Electrical coupling of
cardiac myocyte cell sheets to the heart. Circ Res 98: 573–575, 2006.
24. Ezashi T, Telugu BP, Alexenko AP, Sachdev S, Sinha S, Roberts RM.
Derivation of induced pluripotent stem cells from pig somatic cells. Proc
Natl Acad Sci USA 106: 10993–10998, 2009.
25. Feinberg AW, Feigel A, Shevkoplyas SS, Sheehy S, Whitesides GM,
Parker KK. Muscular thin films for building actuators and powering
devices. Science 317: 1366 –1370, 2007.
26. Fink C, Ergun S, Kralisch D, Remmers U, Weil J, Eschenhagen T.
Chronic stretch of engineered heart tissue induces hypertrophy and
functional improvement. FASEB J 14: 669 –679, 2000.
27. Foldes G, Mioulane M, Wright JS, Liu AQ, Novak P, Merkely B,
Gorelik J, Schneider MD, Ali NN, Harding SE. Modulation of human
embryonic stem cell-derived cardiomyocyte growth: a testbed for studying human cardiac hypertrophy? J Mol Cell Cardiol 50: 367–376, 2011.
28. Furuta A, Miyoshi S, Itabashi Y, Shimizu T, Kira S, Hayakawa K,
Nishiyama N, Tanimoto K, Hagiwara Y, Satoh T, Fukuda K, Okano
T, Ogawa S. Pulsatile cardiac tissue grafts using a novel three-dimensional cell sheet manipulation technique functionally integrates with the
host heart, in vivo. Circ Res 98: 705–712, 2006.
H141
Review
H142
47.
48.
49.
50.
51.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
large-scale tissue restoration on the beating heart after myocardial injury.
Circulation 112: I173–I177, 2005.
Laflamme MA, Murry CE. Heart regeneration. Nature 473: 326 –335,
2011.
Landa N, Miller L, Feinberg MS, Holbova R, Shachar M, Freeman
I, Cohen S, Leor J. Effect of injectable alginate implant on cardiac
remodeling and function after recent and old infarcts in rat. Circulation
117: 1388 –1396, 2008.
Leor J, Aboulafia-Etzion S, Dar A, Shapiro L, Barbash IM, Battler
A, Granot Y, Cohen S. Bioengineered cardiac grafts: a new approach to
repair the infarcted myocardium? Circulation 102: III56 –III61, 2000.
Liau B, Christoforou N, Leong KW, Bursac N. Pluripotent stem
cell-derived cardiac tissue patch with advanced structure and function.
Biomaterials 32: 9180 –9187, 2011.
Loffredo FS, Steinhauser ML, Gannon J, Lee RT. Bone marrowderived cell therapy stimulates endogenous cardiomyocyte progenitors
and promotes cardiac repair. Cell Stem Cell 8: 389 –398, 2011.
Mende U, Eschenhagen T, Geertz B, Schmitz W, Scholz H, Schulte
am Esch J, Sempell R, Steinfath M. Isoprenaline-induced increase in
the 40/41 kDa pertussis toxin substrates and functional consequences on
contractile response in rat heart. Naunyn Schmiedebergs Arch Pharmacol
345: 44 –50, 1992.
Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H,
Ishino K, Ishida H, Shimizu T, Kangawa K, Sano S, Okano T,
Kitamura S, Mori H. Monolayered mesenchymal stem cells repair
scarred myocardium after myocardial infarction. Nat Med 12: 459 –465,
2006.
Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flugel L,
Dorn T, Goedel A, Hohnke C, Hofmann F, Seyfarth M, Sinnecker D,
Schomig A, Laugwitz KL. Patient-specific induced pluripotent stemcell models for long-QT syndrome. N Engl J Med 363: 1397–1409, 2010.
Morritt AN, Bortolotto SK, Dilley RJ, Han X, Kompa AR, McCombe
D, Wright CE, Itescu S, Angus JA, Morrison WA. Cardiac tissue
engineering in an in vivo vascularized chamber. Circulation 115: 353–
360, 2007.
Mummery C, Ward-van Oostwaard D, Doevendans P, Spijker R,
van den Brink S, Hassink R, van der Heyden M, Opthof T, Pera M,
de la Riviere AB, Passier R, Tertoolen L. Differentiation of human
embryonic stem cells to cardiomyocytes: role of coculture with visceral
endoderm-like cells. Circulation 107: 2733–2740, 2003.
Munoz J, Zhou Y, Jarrett HW. LG4 –5 domains of laminin-211 binds
alpha-dystroglycan to allow myotube attachment and prevent anoikis. J
Cell Physiol 222: 111–119, 2010.
Munzel F, Muhlhauser U, Zimmermann WH, Didie M, Schneiderbanger K, Schubert P, Engmann S, Eschenhagen T, Zolk O. Endothelin-1 and isoprenaline co-stimulation causes contractile failure which
is partially reversed by MEK inhibition. Cardiovasc Res 68: 464 –474,
2005.
Nadal-Ginard B, Kajstura J, Anversa P, Leri A. A matter of life and
death: cardiac myocyte apoptosis and regeneration. J Clin Invest 111:
1457–1459, 2003.
Naito H, Melnychenko I, Didie M, Schneiderbanger K, Schubert P,
Rosenkranz S, Eschenhagen T, Zimmermann WH. Optimizing engineered heart tissue for therapeutic applications as surrogate heart muscle.
Circulation 114: I72–I78, 2006.
Norotte C, Marga FS, Niklason LE, Forgacs G. Scaffold-free vascular
tissue engineering using bioprinting. Biomaterials 30: 5910 –5917, 2009.
Okano T, Yamada N, Okuhara M, Sakai H, Sakurai Y. Mechanism of
cell detachment from temperature-modulated, hydrophilic-hydrophobic
polymer surfaces. Biomaterials 16: 297–303, 1995.
Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B,
Pickel J, McKay R, Nadal-Ginard B, Bodine DM, Leri A, Anversa P.
Bone marrow cells regenerate infarcted myocardium. Nature 410: 701–
705, 2001.
Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI,
Taylor DA. Perfusion-decellularized matrix: using nature’s platform to
engineer a bioartificial heart. Nat Med 14: 213–221, 2008.
Papadaki M, Bursac N, Langer R, Merok J, Vunjak-Novakovic G,
Freed LE. Tissue engineering of functional cardiac muscle: molecular,
structural, and electrophysiological studies. Am J Physiol Heart Circ
Physiol 280: H168 –H178, 2001.
Park H, Radisic M, Lim JO, Chang BH, Vunjak-Novakovic G. A
novel composite scaffold for cardiac tissue engineering. In Vitro Cell Dev
Biol Anim 41: 188 –196, 2005.
67. Peng S, Lacerda AE, Kirsch GE, Brown AM, Bruening-Wright A.
The action potential and comparative pharmacology of stem cell-derived
human cardiomyocytes. J Pharmacol Toxicol Methods 61: 277–286,
2010.
68. Pong T, Adams WJ, Bray MA, Feinberg AW, Sheehy SP, Werdich
AA, Parker KK. Hierarchical architecture influences calcium dynamics
in engineered cardiac muscle. Exp Biol Med (Maywood) 236: 366 –373,
2011.
69. Qiao H, Surti S, Choi SR, Raju K, Zhang H, Ponde DE, Kung HF,
Karp J, Zhou R. Death and proliferation time course of stem cells
transplanted in the myocardium. Mol Imaging Biol 11: 408 –414, 2009.
70. Radisic M, Deen W, Langer R, Vunjak-Novakovic G. Mathematical
model of oxygen distribution in engineered cardiac tissue with parallel
channel array perfused with culture medium containing oxygen carriers.
Am J Physiol Heart Circ Physiol 288: H1278 –H1289, 2005.
71. Radisic M, Park H, Martens TP, Salazar-Lazaro JE, Geng W, Wang
Y, Langer R, Freed LE, Vunjak-Novakovic G. Pre-treatment of
synthetic elastomeric scaffolds by cardiac fibroblasts improves engineered heart tissue. J Biomed Mater Res A 86: 713–724, 2008.
72. Radisic M, Park H, Shing H, Consi T, Schoen FJ, Langer R, Freed
LE, Vunjak-Novakovic G. Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds.
Proc Natl Acad Sci USA 101: 18129 –18134, 2004.
73. Radisic M, Yang L, Boublik J, Cohen RJ, Langer R, Freed LE,
Vunjak-Novakovic G. Medium perfusion enables engineering of compact and contractile cardiac tissue. Am J Physiol Heart Circ Physiol 286:
H507–H516, 2004.
74. Reinecke H, Poppa V, Murry CE. Skeletal muscle stem cells do not
transdifferentiate into cardiomyocytes after cardiac grafting. J Mol Cell
Cardiol 34: 241–249, 2002.
75. Reinlib L, Field L. Cell transplantation as future therapy for cardiovascular disease?: A workshop of the National Heart, Lung, and Blood
Institute. Circulation 101: E182–E187, 2000.
76. Revazova ES, Turovets NA, Kochetkova OD, Kindarova LB, Kuzmichev LN, Janus JD, Pryzhkova MV. Patient-specific stem cell lines
derived from human parthenogenetic blastocysts. Cloning Stem Cells 9:
432–449, 2007.
77. Riva E, Hearse DJ. Isolated, perfused neonatal rat heart preparation for
studies of calcium and functional stability. Ann Thorac Surg 52: 987–
992, 1991.
78. Sawa Y, Miyagawa S, Sakaguchi T, Fujita T, Matsuyama A, Saito A,
Shimizu T, Okano T. Tissue engineered myoblast sheets improved
cardiac function sufficiently to discontinue LVAS in a patient with DCM:
report of a case. Surg Today 42: 181–184, 2012.
79. Schaaf S, Shibamiya A, Mewe M, Eder A, Stohr A, Hirt MN, Rau T,
Zimmermann WH, Conradi L, Eschenhagen T, Hansen A. Human
engineered heart tissue as a versatile tool in basic research and preclinical
toxicology. PLoS One 6: e26397, 2011.
80. Sekine H, Shimizu T, Dobashi I, Matsuura K, Hagiwara N, Takahashi M, Kobayashi E, Yamato M, Okano T. Cardiac cell sheet
transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection. Tissue Eng Part A 17:
2973–2980, 2011.
81. Sekine H, Shimizu T, Hobo K, Sekiya S, Yang J, Yamato M,
Kurosawa H, Kobayashi E, Okano T. Endothelial cell coculture within
tissue-engineered cardiomyocyte sheets enhances neovascularization and
improves cardiac function of ischemic hearts. Circulation 118: S145–
S152, 2008.
82. Shimizu T, Sekine H, Yang J, Isoi Y, Yamato M, Kikuchi A,
Kobayashi E, Okano T. Polysurgery of cell sheet grafts overcomes
diffusion limits to produce thick, vascularized myocardial tissues. FASEB
J 20: 708 –710, 2006.
83. Shimizu T, Yamato M, Isoi Y, Akutsu T, Setomaru T, Abe K,
Kikuchi A, Umezu M, Okano T. Fabrication of pulsatile cardiac tissue
grafts using a novel 3-dimensional cell sheet manipulation technique and
temperature-responsive cell culture surfaces. Circ Res 90: e40, 2002.
84. Siedner S, Kruger M, Schroeter M, Metzler D, Roell W, Fleischmann
BK, Hescheler J, Pfitzer G, Stehle R. Developmental changes in
contractility and sarcomeric proteins from the early embryonic to the
adult stage in the mouse heart. J Physiol 548: 493–505, 2003.
85. Soonpaa MH, Field LJ. Survey of studies examining mammalian
cardiomyocyte DNA synthesis. Circ Res 83: 15–26, 1998.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00007.2012 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 16, 2017
52.
PHYSIOLOGICAL ASPECTS OF CARDIAC TISSUE ENGINEERING
Review
PHYSIOLOGICAL ASPECTS OF CARDIAC TISSUE ENGINEERING
98. Weil J, Eschenhagen T, Hirt S, Magnussen O, Mittmann C, Remmers U, Scholz H. Preserved Frank-Starling mechanism in human end
stage heart failure. Cardiovasc Res 37: 541–548, 1998.
99. Wettwer E, Amos GJ, Posival H, Ravens U. Transient outward current
in human ventricular myocytes of subepicardial and subendocardial
origin. Circ Res 75: 473–482, 1994.
100. Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy
M, Henckaerts E, Bonham K, Abbott GW, Linden RM, Field LJ,
Keller GM. Human cardiovascular progenitor cells develop from a
KDR⫹ embryonic-stem-cell-derived population. Nature 453: 524 –528,
2008.
101. Zhang M, Methot D, Poppa V, Fujio Y, Walsh K, Murry CE.
Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death
strategies. J Mol Cell Cardiol 33: 907–921, 2001.
102. Zhao T, Zhang ZN, Rong Z, Xu Y. Immunogenicity of induced
pluripotent stem cells. Nature 474: 212–215, 2011.
103. Zhu WZ, Van Biber B, Laflamme MA. Methods for the derivation and
use of cardiomyocytes from human pluripotent stem cells. Methods Mol
Biol 767: 419 –431, 2011.
104. Zhu WZ, Xie Y, Moyes KW, Gold JD, Askari B, Laflamme MA.
Neuregulin/ErbB signaling regulates cardiac subtype specification in
differentiating human embryonic stem cells. Circ Res 107: 776 –786,
2010.
105. Zimmermann WH. Embryonic and embryonic-like stem cells in heart
muscle engineering. J Mol Cell Cardiol 50: 320 –326, 2011.
106. Zimmermann WH, Didie M, Wasmeier GH, Nixdorff U, Hess A,
Melnychenko I, Boy O, Neuhuber WL, Weyand M, Eschenhagen T.
Cardiac grafting of engineered heart tissue in syngenic rats. Circulation
106: I151–I157, 2002.
107. Zimmermann WH, Eschenhagen T. Embryonic stem cells for cardiac
muscle engineering. Trends Cardiovasc Med 17: 134 –140, 2007.
108. Zimmermann WH, Fink C, Kralisch D, Remmers U, Weil J, Eschenhagen T. Three-dimensional engineered heart tissue from neonatal rat
cardiac myocytes. Biotechnol Bioeng 68: 106 –114, 2000.
109. Zimmermann WH, Melnychenko I, Wasmeier G, Didie M, Naito H,
Nixdorff U, Hess A, Budinsky L, Brune K, Michaelis B, Dhein S,
Schwoerer A, Ehmke H, Eschenhagen T. Engineered heart tissue grafts
improve systolic and diastolic function in infarcted rat hearts. Nat Med
12: 452–458, 2006.
110. Zimmermann WH, Schneiderbanger K, Schubert P, Didie M, Munzel F, Heubach JF, Kostin S, Neuhuber WL, Eschenhagen T. Tissue
engineering of a differentiated cardiac muscle construct. Circ Res 90:
223–230, 2002.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00007.2012 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.246 on June 16, 2017
86. Soonpaa MH, Koh GY, Klug MG, Field LJ. Formation of nascent
intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 264: 98 –101, 1994.
87. Stevens KR, Pabon L, Muskheli V, Murry CE. Scaffold-free human
cardiac tissue patch created from embryonic stem cells. Tissue Eng Part
A 15: 1211–1222, 2009.
88. Studer R, Reinecke H, Vetter R, Holtz J, Drexler H. Expression and
function of the cardiac Na⫹/Ca2⫹ exchanger in postnatal development of
the rat, in experimental-induced cardiac hypertrophy and in the failing
human heart. Basic Res Cardiol 92, Suppl 1: 53–58, 1997.
89. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors. Cell
126: 663–676, 2006.
90. Tanaka H, Namekata I, Nouchi H, Shigenobu K, Kawanishi T,
Takahara A. New aspects for the treatment of cardiac diseases based on
the diversity of functional controls on cardiac muscles: diversity in the
excitation-contraction mechanisms of the heart. J Pharm Sci 109: 327–
333, 2009.
91. Tanaka H, Shigenobu K. Role of beta-adrenoceptor-adenylate cyclase
system in the developmental decrease in sensitivity to isoprenaline in
foetal and neonatal rat heart. Br J Pharmacol 100: 138 –142, 1990.
92. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel
JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from
human blastocysts. Science 282: 1145–1147, 1998.
93. Tiburcy M, Didie M, Boy O, Christalla P, Doker S, Naito H,
Karikkineth BC, El-Armouche A, Grimm M, Nose M, Eschenhagen
T, Zieseniss A, Katschinksi DM, Hamdani N, Linke WA, Yin X,
Mayr M, Zimmermann WH. Terminal differentiation, advanced organotypic maturation, and modeling of hypertrophic growth in engineered
heart tissue. Circ Res 109: 1105–1114, 2011.
94. Tulloch NL, Muskheli V, Razumova MV, Korte FS, Regnier M,
Hauch KD, Pabon L, Reinecke H, Murry CE. Growth of engineered
human myocardium with mechanical loading and vascular coculture.
Circ Res 109: 47–59, 2011.
95. van der Velden J, Klein LJ, van der Bijl M, Huybregts MA, Stooker
W, Witkop J, Eijsman L, Visser CA, Visser FC, Stienen GJ. Force
production in mechanically isolated cardiac myocytes from human ventricular muscle tissue. Cardiovasc Res 38: 414 –423, 1998.
96. Vandenburgh H, Shansky J, Benesch-Lee F, Barbata V, Reid J,
Thorrez L, Valentini R, Crawford G. Drug-screening platform based
on the contractility of tissue-engineered muscle. Muscle Nerve 37:
438 –447, 2008.
97. Vandenburgh HH, Hatfaludy S, Karlisch P, Shansky J. Mechanically
induced alterations in cultured skeletal muscle growth. J Biomech 24,
Suppl 1: 91–99, 1991.
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