Reviews
This Review is part of a thematic series on Stem Cells, which includes the following articles:
Differentiation of Pluripotent Embryonic Stem Cells Into Cardiomyocytes
Derivation and Potential Applications of Human Embryonic Stem Cells
Stem Cells for Myocardial Regeneration
Neural Stem Cells: An Overview
Mesenchymal Stem Cells
Myocyte Death, Growth, and Regeneration in Cardiac Hypertrophy and Failure
Therapeutics and Use of Stem Cells
Toren Finkel, Roberto Bolli, Editors
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Derivation and Potential Applications of Human
Embryonic Stem Cells
Lior Gepstein
Abstract—Embryonic stem cells are pluripotent cell lines that are derived from the blastocyst-stage early mammalian
embryo. These unique cells are characterized by their capacity for prolonged undifferentiated proliferation in culture
while maintaining the potential to differentiate into derivatives of all three germ layers. During in vitro differentiation,
embryonic stem cells can develop into specialized somatic cells, including cardiomyocytes, and have been shown to
recapitulate many processes of early embryonic development. The present review describes the derivation and unique
properties of the recently described human embryonic stem cells as well as the properties of cardiomyocytes derived
using this unique differentiating system. The possible applications of this system in several cardiac research areas,
including developmental biology, functional genomics, pharmacological testing, cell therapy, and tissue engineering, are
discussed. Because of their combined ability to proliferate indefinitely and to differentiate to mature tissue types, human
embryonic stem cells can potentially provide an unlimited supply of cardiomyocytes for cell therapy procedures aiming
to regenerate functional myocardium. However, many obstacles must still be overcome on the way to successful clinical
utilization of these cells. (Circ Res. 2002;91:866-876.)
Key Words: stem cells 䡲 cardiomyocyte differentiation 䡲 cell therapy 䡲 tissue engineering
䡲 myocardial regeneration
O
tific discussion. This interest stems in part from the controversy surrounding the origin of these lines but, more importantly, from the widespread conviction that their availability
will have a major impact on several scientific areas. The
focus of the present review will be to describe the derivation
and unique properties of human ES cells. Particular emphasis
will be placed on describing the potential research and
clinical applications of this new technology in the field of
cardiac research. These applications will be discussed in the
context of the extensive experience gathered over the years
ne of the most promising areas in basic research today
involves the use of stem cells. These unique cells have
the capability to transform and replenish the different tissue
types that make up the body, and they also represent the
fundamental building blocks of human development. In
general, stem cells can be divided into two broad categories:
adult (somatic) stem cells and embryonic stem (ES) cells. The
recent derivation of human ES cell lines from human blastocysts1,2 and human embryonic germ (EG) cell lines from
primordial germ cells3 has aroused intense public and scien-
Original received July 11, 2002; revision received October 1, 2002; accepted October 2, 2002.
From the Cardiovascular Research Laboratory, the Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Rappaport Family
Institute for Research in the Medical Sciences and Rambam Medical Center, Haifa, Israel.
Correspondence to Lior Gepstein, MD, PhD, Cardiovascular Research Laboratory, the Bruce Rappaport Faculty of Medicine, Technion, 2 Efron St,
PO Box 9649, 31096 Haifa, Israel. E-mail [email protected]
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000041435.95082.84
866
Gepstein
Applications of Human Embryonic Stem Cells
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from the analogous murine ES cell model and through a
description of the steps required to fully harness the research
and clinical potential of these cells. The ethical and legal
issues associated with human ES cell research will not be
discussed in the present review but have received thoughtful
coverage elsewhere.4 – 6
Derivation of ES Cells
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All stem cells are defined as having two basic properties:
prolonged self-renewal and the potential to differentiate into
one or more specialized cell type. In adults, a highly regulated
process of stem cell self-renewal and differentiation sustains
tissues with high cell turnover. In recent years, this type of
adult stem cell was also described in tissues that were
originally thought to have relatively limited regenerative
capacity, such as the brain and pancreas.7,8 Although adult
stem cells have been found to be more versatile than
originally believed,9 they typically can differentiate to a
relatively limited number of cell types.
In contrast, cells in the early mammalian embryo have the
potential to contribute to all tissue types in the body, a
property that is termed pluripotency. After fertilization, at the
blastocyst stage, a hollow sphere of cells is formed that
contains an outer cell layer and an inner cluster of cells
termed the inner cell mass (ICM, Figure 1). Whereas the outer
cells become the trophectoderm and subsequently give rise to
the placenta and other supporting tissues, the ICM cells will
ultimately create all tissues in the body, as well as nontrophoblast structures that support the embryo and are therefore
truly pluripotent.10
The term ES cells originated from the isolation in 1981 of
pluripotent stem cell cultures from mouse blastocysts by
Evans and Kaufman11 and independently by Martin.12 The ES
cells were discovered to be capable of self-renewal and
prolonged undifferentiated proliferation in culture. However,
because of their origin in the early embryo, ES cells differed
from other stem cells in their ability to retain the potential to
generate derivatives of all three germ layers. Interestingly,
although these cells could proliferate indefinitely in the
undifferentiated state in cultures, the embryonic cells from
which they were derived (the ICM cells), in vivo, are
normally short-lived and soon disappear from the developing
embryo as they give rise to specialized progenitor cells of the
three germ layers.
Given the large impact that the development of the murine
ES cell lines has had on several research fields in the last 20
years, it is not surprising that significant efforts have been
invested in the derivation of human pluripotent stem cell
lines. To date, three pluripotent cell types have been established from human tissue: human embryonic carcinoma (EC)
cells,13,14 human EG cells,3 and human ES cells.1,2
Human EC cell lines and their murine counterparts were
the first pluripotent stem cell lines to be established. They
were derived from the undifferentiated stem cell component
of germ cell tumors. The EC clones could be expanded
continuously in culture but could also differentiate to produce
derivatives of all three germ layers either in vitro or through
teratocarcinoma formation. Compared with human ES cells,
however, these cells seem to have less differentiating capac-
Figure 1. Early embryonic mammalian development and derivation of the ES cell lines. At the blastocyst stage (5 days after
fertilization), the embryo is composed of the trophectoderm and
the ICM, which eventually will give rise to all tissue types in the
embryo. The ES cell lines were generated from the ICM. These
cells were isolated by immunosurgery and plated on the MEF
feeder layer. The resulting colonies were propagated and
expanded. ES cells can be propagated in the undifferentiated
state while being cultured on top of the MEF feeder layer. When
they are removed from the feeder layer and cultivated in suspension as 3D cell aggregates (EBs), the ES cells differentiate
into specialized cells, including neuronal, hematopoietic, skeletal
muscle, smooth muscle, and cardiac tissue.
ity, and they are usually aneuploid and, therefore, not suitable
for clinical applications.15 Pluripotent human and mouse EG
lines were derived from primordial germ cells in the genital
ridges of the developing embryos, typically at 5 to 9 weeks
after fertilization in humans,3 and were also shown to be
pluripotent.
The origin of human ES cells, in contrast to EC and EG
cells, is from the preimplantation embryo. These cell lines
were derived from the ICM cells of human blastocysts,
produced by in vitro fertilization for clinical purposes and
donated by individuals after informed consent. The human ES
cell lines were created in a manner similar to that of
mouse11,12 and rhesus16 ES cells. In this process, the outer
trophectoderm layer of the blastocyst was selectively removed using specific antibodies (immunosurgery), and the
ICM cells were isolated and plated on a mitotically inactivated mouse embryonic fibroblast (MEF) feeder layer (Figure
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1). Cells from the periphery of the colonies that formed were
mechanically isolated and replated in the same fashion until
homogenous colonies appeared. These colonies were selected, passaged, and expanded for the creation of the ES cell
lines.
The human ES cells were demonstrated to fulfill all the
criteria defining embryonic stem cells, namely, derivation
from the preimplantation embryo, prolonged undifferentiated
proliferation in culture under special conditions, and the
capacity to form derivatives of all three germ layers.1 In
addition, the human ES lines have also been shown to keep a
stable diploid karyotype and to continuously express a high
level of telomerase activity during long-term propagation in
culture.1,17
The pluripotency of ES cells can be established traditionally using three different approaches. Mouse ES cells can be
retransferred into early mouse embryos, where they eventually give rise to all somatic cells of the chimeric embryo,
including the germ cells.18 Such a test cannot be applied to
human ES cells for obvious ethical reasons. The second
approach relates to the demonstration that ES cells can
differentiate to generate derivatives of all three germ layers in
vivo. When human ES cells were injected into immunodeficient mice, they formed benign teratomas containing advanced differentiated tissue types representing all three germ
layers.1,17 The third approach establishes ES pluripotency
during in vitro differentiation. Both mouse and human ES
cells, when removed from the MEF feeder layer and allowed
to differentiate, could form 3D cell aggregates, termed
embryoid bodies (EBs), that contained tissue derivatives of
endodermal, ectodermal, and mesodermal origin.19,20
The originally described human ES cell lines were not
clonally derived from a single cell; therefore, their pluripotency could theoretically be attributed to a population of cells.
In further studies, Amit et al17 produced single-cell clones
from the human ES cell lines. This was accomplished by
plating individual cells derived from the parental line under
direct observation on individual wells containing the MEF
feeder. Although the cloning efficiency of the human ES cells
was relatively poor, a severalfold increase was observed
when serum-free medium supplemented with basic fibroblast
growth factor (FGF) was used. The human ES cell-derived
clones retained all the properties of the parental line, including prolonged undifferentiated proliferation with a stable
karyotype, expression of high levels of telomerase, and the
ability to generate teratomas after in vivo transplantation to
immunodeficient mice.
In Vitro Differentiation
One of the most fascinating and important aspects of ES cell
lines is their ability to differentiate in vitro, via precursor
cells, into terminally differentiated somatic cells of all tissue
types. The mouse ES cells were shown to differentiate in vitro
into a variety of cell types, including cardiomyocytes,19
skeletal and smooth muscle cells,21,22 neuronal and glial
cells,23,24 hematopoietic progenitor cells,19 adipocytes,25 endothelial cells,26 pancreatic islet cells,27 and several other
tissue types.28 Ever since the initial report of derivation of the
human ES cell lines, a variety of studies have also established
in vitro spontaneous and directed differentiation systems to
several lineages, including cardiac tissue,29 neuronal tissue,30,31 ␤ islet pancreatic cells,32 hematopoietic progenitors,33 and endothelial cells.34
The most common method used for in vitro differentiation
is to remove the ES cells from the feeder layer and to cultivate
them into 3D cell aggregates, termed EBs (Figure 1). This
aggregation step is required for the differentiation of most
cell lineages (except neurogenesis), and it is believed that the
cell interactions within the 3D structure are important for
differentiation. Different protocols have been used in the
murine ES cells for such cultivation, including the “mass
culture” technique,19 cultivation in methylcellulose, or the
“hanging drops” technique.35 In the latter technique, a definite number of mouse ES cells (usually 400) are cultivated in
hanging drops for 2 days.36 The EBs are then collected and
further cultivated for an additional 5 days in suspension and
then plated on coated culture dishes for further differentiation. Among other cell types, cardiomyocyte tissue could be
identified in the outgrowth of the plated murine EBs as
clusters of spontaneously beating areas.36
Similar to the mouse model, human ES cells, when
removed from the MEF feeder layer and cultivated in
suspension, begin to differentiate by forming EBs, some of
which develop to cystic EBs.37 Although these EBs were
found to be somewhat less organized than the mouse-derived
EBs, they characteristically displayed regional expression of
embryonic markers specific to different lineages of ectodermal, mesodermal, and endodermal origin.20
More recently, we have used methodologies slightly different from those reported in the mouse model to generate a
reproducible spontaneous cardiomyocyte-differentiating system from human ES cells.29 Human ES cells were dissociated
into small clumps of 3 to 20 cells and grown in suspension for
7 to 10 days, where they formed EBs. The EBs were then
plated on gelatin-coated culture dishes and observed microscopically for the appearance of spontaneous contraction.
Rhythmically contracting areas appeared at 4 to 22 days after
plating in 8.1% of the EBs.
Several lines of evidence confirmed the cardiomyocyte
phenotype of these contracting areas (Figure 2). Cells isolated
from the beating areas expressed the cardiac transcription
factors GATA4 and Nkx2.5 and cardiac-specific genes, such
as cardiac troponin I and T, atrial natriuretic peptide (ANP),
and atrial and ventricular myosin light chains (MLCs).
Immunostaining studies demonstrated the presence of the
cardiac-specific sarcomeric proteins myosin heavy chain,
␣-actinin, desmin, and cardiac troponin I as well as ANP
(Figure 2A). Electron microscopy revealed varying degrees
of myofibrillar organization, consistent with the ultrastructural properties reported for early-stage cardiomyocyte (Figure 2B).
The human ES-derived cells also exhibited a functional
phenotype of early-stage human cardiomyocytes, including
electrical activity, calcium transients, and chronotropic response to adrenergic agents (Figures 2C and 2D). More
recently, we have demonstrated that this system is not limited
to the differentiation of isolated cardiomyocyte cells but
rather that a functional cardiomyocyte syncytium is generated
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Figure 2. Structural and functional characterization of the human ES cell– derived cardiomyocytes. A, Immunostaining
with anti-cardiac ␣/␤-MHC, antisarcomeric ␣-actinin, anti-cardiac troponin I (anti-cTnI), anti-desmin, and antiANP antibodies. B, Transmission
electron micrograph showing an early
cardiomyocyte ultrastructural pattern. C,
Extracellular electrophysiological recordings from the beating EB. D, Calcium
transients as determined by fura 2 fluorescence. E, Electrical activation map of
a contracting EB generated by a microelectrode array mapping technique that
simultaneously records electrograms
from 60 electrodes. Colors represent
local activation times, with activation initiating from the red area and propagating
to the rest of the tissue. Figure was
adapted from 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. 2001;108:407– 414,
by permission.
with synchronous action potential propagation and pacemaker activity38 (Figure 2E).
Differences Between Human and
Murine Models
Although experience with human ES cells is relatively
limited, important differences can already be noted when
these cells are compared with the murine model.39 Human ES
cells form relatively flat and compact colonies that can be
easily dissociated into single cells with conventional mechanical and enzymatic techniques. The population-doubling time
of human ES cells (36 hours) is significantly longer than that
of mouse ES cells (12 hours). In addition, the two sources
differ in some of their antigenic phenotypes. For example,
human ES cells express stage-specific embryonic antigen
(SSEA)-3 and SSEA-4, which are not expressed in mouse ES
cells but lack SSEA-1, which is expressed in the latter
model.1
One of the most important differences between the two
models lies in their in vitro culturing requirements. In contrast
to the mouse model in which the undifferentiated propagation
of ES cells can be achieved by the administration of leukemia
inhibitory factor, the human ES cells require the presence of
a feeder layer. This is achieved by propagating the cells on
top of the MEF feeder layer in the presence of serum or serum
replacement supplemented with basic FGF. More recently,
the undifferentiated proliferation of the human ES cells was
also described in feeder-free settings using conditioned media
from the MEF feeder layer40 and also by maintaining the ES
cells on a human feeder cell layer.41
In addition to the differences observed between the human
and mouse ES cell lines, there are also important variations in
the in vitro cardiomyocyte differentiation properties of the
lines. Compared with murine cells, human ES cells differentiate in vitro into cardiomyocytes at a slower rate. Furthermore, whereas transmission electron microscopy revealed a
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reproducible progressive process of ultrastructural development from an irregular myofilament distribution to a more
mature sarcomeric organization during ⬍2 weeks in the
mouse model,36,42 this process was more heterogeneous,
lasted longer, and did not reach the same level of maturity in
the human model. These findings are not surprising considering that there are differences in the length of the gestational
periods and that the in vivo formation of the human heart
takes place during the first 35 days, in contrast with 12 days
in the mouse. These differences may represent differences
between the species, differences between the cell lines, or
differences in the in vitro culturing techniques.
Implication for Basic Research and
Clinical Applications
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Because of the outstanding potential demonstrated by mouse
ES lines, the introduction of human ES cell lines may hold
important research and clinical applications. Specifically,
important topics related to cardiac research may include the
following: (1) investigation of the mechanisms involved in
early human cardiac lineage commitment, differentiation, and
maturation; (2) derivation of a unique in vitro model to study
human cardiac tissue; (3) functional genomics, drug and
growth factor discovery, drug testing, and reproductive toxicology; and (4) development of cell-based therapies and
tissue-engineering strategies.
In contrast to the fairly well-characterized process of the
morphogenic transformation of the primitive heart into the
4-chambered structure,43 the inductive clues that lead to
specification and terminal differentiation of cardiomyocytes
are somewhat less known. Although organogenesis or significant tissue organization does not occur within the EB model,
valuable information can be gathered regarding the process
involved in lineage commitment and differentiation. In fact,
in vitro differentiation within the EB model system may
provide a number of advantages over comparable approaches
in the whole embryo. First, it provides access to the population of early precursor cells that are difficult if not impossible
to identify in vivo. Second, it could allow the study of
targeted mutations of genes that may be lethal in vivo but can
be studied in vitro. These advantages are even more important
for human embryology because of the limited access to
early-stage human tissue.
Data derived from amphibian and chick embryos and, more
recently, from mouse embryos have suggested that signals
emanating from the primitive endoderm in the early embryo
may be involved in the processes of cardiomyocyte induction
of the precursor cells in the adjacent anterior mesoderm.44,45
Recent studies have also identified some of the growth factors
that regulate cardiomyogenic induction. These include molecules that promote cardiomyogenesis (bone morphogenetic
proteins [BMPs], FGFs, and inhibitors of the Wnt family of
morphogens) as well as factors that inhibit the process (Wnt
family of morphogens, noggin, and chordin).46 – 48
Evidence exists that the above-mentioned findings may
also be relevant to ES cell differentiation. Coculturing experiments of undifferentiated P19 EC cells, mouse ES cells, and
human ES cells with END-2, an endoderm-like cell line,49
promoted their differentiation into immature cardiomyo-
cytes.50,51 Similarly, mouse EBs depleted of primitive
endoderm or parietal endoderm did not develop beating
cardiomyocytes. Several lines of evidence also indicate a role
for extracellular matrix proteins in the regulation of ES cell
cardiac differentiation.52 For example, studies on ␤1-integrin–
deficient mouse ES cells demonstrated impaired cardiac
differentiation with delayed expression of cardiac-specific
genes and action potentials and impaired sarcomeric organization. Interestingly, these studies also implicate an important
role for Wnt-1 and -4 pathways on cardiogenesis, in a manner
similar to that of the in vivo findings.52
The in vitro formation of cardiomyocytes within the EB
also provides a unique tool for the investigation of early
cardiomyogenesis. Detailed ultrastructural, immunohistochemical, molecular, and electrophysiological studies have
shown that the developmental stages of the mouse ES
cell– derived cardiomyocytes in vitro recapitulate those of the
in vivo murine heart.36,53– 60 During in vitro cardiomyocyte
differentiation, a developmentally controlled expression of
cardiac-specific genes was noted.54 The mesoderm-specific
genes, such as BMP-4, were expressed initially, followed by
gene expression of cardiac-specific transcription factors
(Nkx2.5) and structural cardiac proteins, with the chamberspecific genes, such as MLC-2V, expressed last.
The formation of cardiomyocytes within the EB may also
provide a unique in vitro tool for investigation of the
physiological processes involved in early cardiomyocyte
differentiation and maturation. The advent of the murine ES
cell model has thus provided important insights into the early
stages of development of excitability and electromechanical
coupling, including patterns of gene expression, myofibrillogenesis, ion channel development and function, calcium
handling, receptor development, and the signal machinery
involved in these processes.36,53 Moreover, because of the
relative ease in genetically manipulating the ES cells, reporter
gene constructs may be used in conjunction with early
cardiac-specific promoters to identify and study early-stage
cardiac precursor cells before the initiation of spontaneous
contraction.61,62
Detailed electrophysiological studies of cardiomyocytes
within the developing murine EBs have revealed a developmental cascade of ion channel expression and modulation.36,63 The noncontracting precursor cells already displayed
voltage-dependent L-type Ca2⫹ channels at very low densities. Cardiomyocytes of a very early differentiating stage had
an action potential that was generated by only two main types
of ion channels, the L-type Ca2⫹ channel and the transient K⫹
channel. Additional ion channels, such as the voltagedependent Na⫹ channel, delayed outward rectifying K⫹ channel, inward rectifying K⫹ channel, muscarinic activated K⫹
channel, and pacemaker channel, were demonstrated, only in
more differentiated cardiomyocytes. The increased number of
different ion channels caused a diversification of cardiac
phenotypes, with cardiomyocytes in late-stage EBs displaying a ventricle-like, atrium-like, and Purkinje-like
phenotype.58
Another important property of the human ES cell– differentiating system is the ability to reproducibly provide differentiated nontransformed cardiomyocytes for long term in
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vitro assessment of cardiac tissue. Although the heart has
been thoroughly investigated in its intact form, only a small
number of in vitro models are currently available for the study
of its structural and functional properties during normal
physiological and pathological states. These models include a
number of primary cultures, which may be limited by their
relatively short-term availability and by the lack of a similar
human model. The ability of human ES cells to provide in
vitro cardiomyocyte tissue for long-term assessment may also
prove invaluable for drug discovery, drug screening, and
toxicity testing. Furthermore, by use of the differentiation of
the murine ES cells to cardiomyocytes, a standardized in vitro
model (the so-called embryonic stem cell test) has already
been derived to analyze the embryotoxic effects of chemical
compounds.64
Another possible important application of the human ES
cell– differentiating system is to determine the function of
novel genes during differentiation and also in terminally
differentiated cells. Because the cardiomyocytes in this model
can be derived from a clonal precursor, genetic manipulation
may be performed with relative ease. This theoretically may
provide a unique system to assess the function of human
genes through “loss of function” studies or “gain of function”
approaches.
Developing Transplantation Strategies for
Myocardial Regeneration
The most exciting and frequently discussed application of ES
cells is in cell replacement therapy, involving the replacement
of diseased, absent, or malfunctioning tissue. The adult heart
has a limited regenerative capacity; therefore, any significant
cell loss, such as that which occurs during a large myocardial
infarction, is mostly irreversible and may lead to progressive
deterioration in ventricular function and to the development
of heart failure. Congestive heart failure is currently a
growing epidemic affecting ⬎5 million Americans, with
400 000 new cases each year.65 Despite advances in pharmacological, interventional, and surgical therapeutic measures,
the prognosis for patients with this disease remains poor.
With a chronic lack of donors limiting the number of patients
who can benefit from heart transplantations, development of
new therapeutic paradigms for heart failure has become
imperative.
One of the most attractive therapeutic approaches for the
treatment of heart failure may be the development of myocardial regeneration strategies aiming to replace the dysfunctional myocardium with new contractile tissue.66 A number of
myogenic cells have been suggested as potential sources for
tissue grafting, including skeletal myoblasts,67– 69 fetal cardiomyocytes,70,71 smooth muscle cells,72 murine embryonic stem
cells,73 and bone marrow– derived stromal74,75 and hematopoietic stem cells.76 Recent animal studies have shown that
cells derived from all sources may survive, differentiate, and
even improve cardiac function after cell grafting.
Although a number of myocyte preparations have been
used in the aforementioned studies, the inherent electrophysiological, structural, and contractile properties of cardiomyocytes strongly suggest that they may be the ideal donor cell
type. Consequently, much effort has been spent over the years
Applications of Human Embryonic Stem Cells
871
in assessing the ability of fetal, neonatal, or adult cardiomyocytes to survive and improve myocardial performance in
animal models. In the pioneering work of Soonpaa et al,70
fetal cardiomyocytes transplanted into mice hearts were
demonstrated to survive, align with host cells, and form
cell-to-cell contacts with host myocardium. The same group
also reported on the formation of stable fetal grafts in the
myocardium of dystrophic mice and dogs.77
Other studies have demonstrated that fetal or neonatal
cardiomyocytes can also be engrafted into infarcted or cryoinjured hearts. An interesting report also suggests that
early-stage cardiomyocytes (fetal and neonatal cells) may
serve as better candidates than adult cardiomyocytes because
of their superior survival rate after transplantation in healthy,
injured, and scarred rat hearts.78 More recently, it was
demonstrated in a rat model of chronic infarction that these
cells could survive and improve cardiac function for up to 6
months.79 Cardiomyocyte cell transplantation has been associated with smaller infarcts80 and has been shown to prevent
cardiac dilatation and remodeling after myocardial infarction81 and to improve ventricular function.82 The mechanism
underlying this functional improvement is not clear. A
number of potential mechanisms have been proposed, including direct contribution to contractility by the transplanted
myocytes, attenuation of the remodeling process by changing
the elastic properties of the scar, and improvement in the
function of viable myocardial tissue by the induction of
angiogenesis.
Despite these encouraging results, the clinical utility of
fetal cardiomyocyte transplantation is significantly hampered
by the inability to obtain human fetal cardiomyocytes in
sufficient numbers for practical and ethical reasons. In addition, fetal and neonatal cells are prone to immune rejection
after transplantation, and they have limited proliferative
potential in vitro and are relatively sensitive to ischemic
injury. Consequentially, significant cardiomyocyte cell death
has been reported after cell transplantation, limiting the
amount of new myocardium that can be formed.83,84
The derivation of human pluripotent ES cell lines offers a
number of potential advantages for cell therapy procedures.
Because of their in vitro ability to be propagated in mass and
to differentiate into cardiomyocytes with the characteristic
electrophysiological, structural, and contractile properties,29
ES cells can potentially provide an unlimited number of cells
for transplantation. Furthermore, specialized subtypes of
cardiomyocytes with different phenotypes (eg, atrial, ventricular) can be differentiated in vitro from ES cells58 and can be
tailored to specific procedures. The ES cell cultures could
lend themselves to extensive characterization and genetic
engineering to promote desirable characteristics, such as
resistance to ischemia and apoptosis and improved contractile
function.
Despite the exciting advances in human ES research, much
basic work is still required, and several obstacles remain to be
overcome before this technology can enter any serious
clinical practice. Specifically, a number of important milestones need to be achieved: (1) Strategies need to be developed for directing in vitro differentiation to a specific lineage.
(2) Selection protocols must be devised to generate a pure
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population of cells of a specific lineage (cardiac) or even of
a specific cell type (eg, ventricular, atrial). (3) Culturing
techniques need to be upscaled for mass production, yielding
clinically relevant quantities of cardiomyocytes. (4) A transplantation technique should be developed, and important
issues regarding the in vivo properties of the graft should be
resolved. (5) Antirejection regimens need to be developed.
Directed Differentiation and Cardiomyocyte
Enrichment Strategies
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Although both human and mouse ES cells have been demonstrated to differentiate spontaneously in vitro to cardiomyocyte tissue using the EB system, the cues involved in this
process are still largely unknown. Moreover, cardiomyocyte
cells account for only a minority of the cells within the EBs,
and spontaneously contracting areas are not observed in all
EBs, even less so in the human model.
Strategies for directed differentiation may follow development in the understanding of the biological processes involved in early cardiomyocyte differentiation or may follow
a trial-and-error approach using different growth factors,
transcription factors, feeder layers,51 and physical perturbations.85 Some evidence suggests a possible role for a number
of soluble factors in promoting cardiomyocyte differentiation.86 These include members of the transforming growth
factor-␤ family, retinoic acid,87 leukemia inhibitory factor,88
and other factors when provided at the appropriate timing and
dosage. However, the extent to which these different factors
may actually promote cardiogenesis is still unknown. A
similar strategy may be applied to promote differentiation of
a specific sublineage phenotype. Recent studies have identified a number of transcriptional factors and growth factors
that may play a role in the specification of atrial, ventricular,
and conduction system cells.89,90
Evidence also exists indicating that undifferentiated human
ES cells and their more differentiated derivatives express
receptors for various growth factors.91 Hence, supplementation of the human ES culture medium with 8 different growth
factors altered the expression profile of an array of tissuerestricted genes. Although some of these growth factors were
shown to increase differentiation to mesoderm, ectoderm, or
endoderm derivatives, none of the growth factors directed
differentiation exclusively to one cell type.
Cell-Selection Strategies
Although differentiation toward a specific lineage may be
enhanced somewhat by the addition of growth factors, the
degree of purity that will be achieved will probably be
insufficient for clinical purposes. Besides the obvious need
for larger number of cardiomyocytes, a selection process may
be required to avoid the presence of other cell derivatives and
to ensure the absence of remaining pluripotent stem cells in
the graft. The latter may be crucial to prevent the possible
generation of ES cell–related tumors, such as teratomas. A
similar strategy may also be necessary to select for the
specific cardiac cell types (eg, atrial, ventricular, or pacemaking cells) required for different cell therapy strategies.
Given the heterogeneous cell mixture within the EB,
derivation of a relatively homogenous cell population will
ultimately depend on the selection of a specific cell type from
the mixed population of cells. An elegant and relatively
simple approach to generate pure cardiomyocyte cultures was
first demonstrated by Field’s group (Klug et al73) in the
murine ES model. This approach is based on the use of a
tissue-specific promoter to drive a selectable marker, such as
an antibiotic resistance gene. Using this approach, Klug et al
generated, by stable transfection, an ES cell line carrying a
fusion gene composed of the ␣-cardiac myosin heavy chain
(␣-MHC) promoter and a cDNA encoding aminoglycoside
phosphotransferase (neoR). The ES cells were then allowed to
differentiate in vitro and were subjected to selection with the
appropriate antibiotic (G418). Using the selection process
during in vitro differentiation, the authors have shown that
⬎99% pure cardiomyocyte cultures could be generated. The
selected cardiomyocytes were further demonstrated to form
stable grafts after transplantation into adult dystrophic mice
hearts.
Interestingly, a similar enrichment strategy has also been
successfully attempted in murine ES cells to produce purified
cultures of neurons92 and insulin-secreting pancreatic ␤
cells27 using the appropriate promoters.
An alternative approach could involve the introduction of a
gene construct containing a tissue-specific promoter/enhancer
controlling the expression of a green fluorescence protein
gene. Selection of the desired cells can then be performed
using fluorescence-activated cell sorting. Using this approach, Muller et al93 transfected murine ES cells with a
construct encoding a cytomegalovirus enhancer and a
ventricle-specific (MLC-2V) promoter driving the green fluorescence protein product. The use of Percoll gradient centrifugation and subsequent fluorescence-activated cell sorting
yielded 97% pure cardiomyocyte fractions.
Both of the above-mentioned approaches would require the
development of efficient gene transfer methodologies for
human ES cells. In a recent report, Eiges et al94 reported on
the establishment of human ES cell–transfected clones carrying a marker for undifferentiated cells. In that report, various
transfection methodologies were used, and their efficiency in
introducing DNA into human ES cells was compared.
Upscaling Methodologies and Strategies for
Prevention of Immune Rejection
In addition to the conceptual milestones that remain to be
achieved for the development of any clinical application,
several technical obstacles also need to be overcome. These
include the development of mass production methods to yield
a clinically relevant number of cells, replacement of animalrelated products used during human ES culturing, qualitycontrol testing, and development of methods for storing and
shipping the cells, such as cryopreservation.
A major barrier for the possible use of human ES cells in
cell transplantation strategies is the generation of sufficient
numbers of cardiomyocytes. It is estimated that a typical
myocardial infarction that induces heart failure kills ⬇1
billion cardiomyocytes: (⬇20 million myocytes/g)⫻(⬇200
g/left ventricle)⫻(⬇25% of left ventricle infarcted). Generation of this number of cells can be achieved by increasing the
initial number of ES cells used for cardiomyocyte differenti-
Gepstein
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ation, by increasing the percentage of cells differentiating to
the cardiac lineage (using the enrichment protocols discussed
above), by increasing the ability of the cells to proliferate
after cardiomyocyte differentiation, and by upscaling the
entire process using bioreactors and related technologies.
Another major consideration and significant obstacle for
the possible clinical utilization of the ES cells is the expected
immune-mediated rejection of the ES cell– derived grafts.
Although a detailed discussion of this issue is beyond the
scope of the present review, we will briefly discuss some of
the strategies suggested to deal with this problem.
The first question to be contended with is as follows:
Precisely how immunogenic are tissues derived from ES
cells? In a recent report,95 human ES cells were shown to
express low levels of MHC class I proteins. This expression
was only moderately increased during in vitro and in vivo
differentiation but was significantly augmented after treatment of the cells with interferon-␥. In contrast, MHC class II
antigens and the ligands for NK cell receptors were not
expressed on the surface of the ES cells or their derivatives.
The absence of MHC class II antigens on human ES cells and
their cardiomyocyte derivatives may be important because
cells expressing these proteins, such as B cells, macrophages,
dendritic cells, and endothelial cells, are believed to be highly
immunogenic. This may provide an inherent immune advantage to human ES cell– derived grafts, which could possibly
require milder immunosuppressive regimens. In addition,
strategies aimed at reducing the mass of alloreactive T cells
are being developed, and these and other novel therapies with
particular relevance to the anticipated immune response
mounted against ES cell– derived cell transplants will probably be used.96
Other approaches for reducing graft rejection may be the
generation of a panel of ES cell lines to allow matching of the
major histocompatibility determinants and genetically modifying the ES cells to reduce their immunogenicity. The latter
can be achieved by engineering a “universal cell donor” by
knocking out the major histocompatibility complexes97 or by
inserting or deleting other genes that can modulate the
immune response.98,99
The concept of hematopoietic chimerism was demonstrated in bone marrow–transplanted patients receiving a
solid organ transplant from the same donor, in which no
immune suppression was required to prevent rejection.100 The
same concept theoretically can be applied also to ES cell–
derived transplantation using the same ES cell line to derive
both lymphohematopoietic stem cells and cardiomyocyte
tissue. Tolerance can then be induced by initially achieving
hematopoietic chimerism followed by cardiomyocyte cell
transplantation.
Derivation of a human ES cell line specifically for each
patient with the use of somatic nuclear transfer technology is
another approach that has been suggested to overcome tissue
rejection. Although the feasibility of this scenario has already
been demonstrated in a bovine population101 in which immunocompatible tissue grafts were generated, a similar therapeutic cloning strategy in humans would be exceptionally
controversial. In addition, the poor availability of human
oocytes and the technical difficulties involved in the process
Applications of Human Embryonic Stem Cells
873
of the creation of each ES cell line makes this approach
unlikely to become a routine clinical procedure.
In Vivo Transplantation
Although changes in the remodeling processes, prevention of
ventricular dilatation, and improvement in the ventricular
diastolic properties have been reported after the transplantation of a variety of cell types into the infarcted region, systolic
augmentation would depend on the functional integration
between graft and host cardiomyocytes. This would require
the long-term survival of the grafted cells, the presence of a
critical tissue mass, the appropriate alignment of the donor
cells, and their structural and functional integration with the
host tissue.
Several questions and issues remain to be addressed in this
area.66 First, the size of the cell graft may have important
applications for its ultimate success in improving the ventricular mechanical function. Cell death occurring after engraftment is believed to have a major negative impact on graft
size.83,84 Hence, cell survival after transplantation may depend on adequate vascularization of the graft, which may
require additional revascularization procedures or induction
of angiogenesis. In that respect, the ability to genetically
manipulate the ES cell derivatives may be used to generate
cell grafts that are more resistant to ischemia or apoptosis,
that display larger proliferative capacity, that can secrete
angiogenic growth factors, or that may be coupled with ES
cell– derived endothelial progenitor cells. Additional factors
that remain to be determined in future studies include the
ideal nature of the graft (individual cells, small cell clumps,
or combined with scaffolding biomaterials), the appropriate
delivery method (epicardial, endocardial, or the coronary
circulation), and the timing of cell delivery relative to the
timing of the infarct.66
The ability to generate potentially unlimited numbers of
cardiomyocytes ex vivo from human ES cells may bring a
unique value also to tissue-engineering approaches. This new
discipline combines functional cells with 3D polymeric scaffolds to create tissue substitutes.102 These biodegradable
scaffolds can be engineered to provide adequate biomechanical support for the cell graft, to control graft shape and size,
and to promote angiogenesis. Recently, scaffolds that may
allow mechanical contraction have also been described.103
An important aspect related to the possible utilization of
ES cell– derived cardiomyocytes for cell transplantation strategies relates to the safety of these procedures. A major safety
concern is the possible development of ES cell–related
tumors. As described above, injection of undifferentiated ES
cells into immunodeficient mice has resulted in the generation of teratomas.1 Hence, as ES cells are coaxed to differentiate in vitro to terminally differentiated cardiomyocytes,
efforts need to be made to ensure that the cell graft is depleted
of any undifferentiated stem cells before transplantation. In
that respect, studies involving in vivo transplantation of
differentiated ES cell derivatives have failed to show any
evidence of teratoma or the presence of any other ES
cell– derived tumor.30,31,73
A second major concern relates to the development of
cardiac arrhythmias after cell transplantation. Besides the
874
Circulation Research
November 15, 2002
known increased risk for malignant ventricular arrhythmias in
patients with reduced ventricular function, cell transplantation may modify the electrophysiological properties of the
scar and theoretically may change (increase or decrease) the
propensity for development of arrhythmias.
Summary
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During the last 20 years, mouse ES cells have revolutionized
many areas of modern biology. The derivation in 1998 of
human ES cell lines was accompanied by significant enthusiasm but also by major public debate. Although research
using human ES cells is still at its “embryonic” stage,
important research accomplishments have already been
achieved. These include the derivation of a number of ES cell
lines and clones, verification of the pluripotent nature of
human ES cell lines both in vivo and in vitro by creation of
teratomas and EBs, respectively, and the establishment of in
vitro differentiating systems to generate variable tissue types,
including cardiomyocytes.
In addition, basic methodologies for large-scale cultures
have been developed, initial functional and morphological
studies of ES cell– derived cardiomyocytes have been performed, and the initial groundwork for genetic manipulation
of these lines has been established. On the basis of these
initial achievements, together with the extensive experience
gathered from the murine model, we can predict that human
ES cells may be useful in the future in a variety of cardiovascular research areas, including human developmental
biology, functional genomics, pharmacological testing, tissue
engineering, and gene- and cell-based therapies. Nevertheless, important knowledge is still lacking in many of these
areas, much basic work is required, and several obstacles
need to be overcome before any clinical breakthroughs can be
expected.
Acknowledgment
This study was supported in part by the Israel Science Foundation.
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Derivation and Potential Applications of Human Embryonic Stem Cells
Lior Gepstein
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Circ Res. 2002;91:866-876
doi: 10.1161/01.RES.0000041435.95082.84
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