Critical Reviews in Oncology/Hematology 65 (2008) 54–80
Human embryonic stem cells: Current technologies
and emerging industrial applications
Caroline Améen a , Raimund Strehl a , Petter Björquist a ,
Anders Lindahl b , Johan Hyllner a , Peter Sartipy a,∗
b
a Cellartis AB, Arvid Wallgrens Backe 20, 413 46 Göteborg, Sweden1
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine,
Sahlgrenska University Hospital, 413 45 Göteborg, Sweden
Accepted 27 June 2007
Contents
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2.
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Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pluripotent hES cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Establishment of hES cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Cultivation of hES cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Spontaneous differentiation of hES cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4. Characterization and quality control of hES cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5. Xenofree derivation and cultivation of hES cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Derivation of functional cells from hES cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Embryogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1. Hepatogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2. Stem cell differentiation to hepatic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.3. Differentiation of hES cells to hepatic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Cardiomyocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1. Cardiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2. ES cell differentiation to cardiac myocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.3. Characteristics of hES-CM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.4. Cell selection and enrichment strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.5. Cardiac progenitor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Applications of hES cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Human ES cells in regenerative medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.1. The heart as a regenerative organ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.2. Human ES cells and cell therapy for cardiac regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.3. Concerns with hES cells in cell therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.4. Activation of endogenous stem cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Human ES cells for use in drug discovery and toxicity testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1. Developmental toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.2. Applications for hepatocytes derived from hES cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.3. Applications for cardiomyocytes derived from hES cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Corresponding author. Tel.: +46 31 7580930; fax: +46 31 7580910.
E-mail address: [email protected] (P. Sartipy).
www.cellartis.com.
1040-8428/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.critrevonc.2007.06.012
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Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract
The efficiency and accuracy of the drug development process is severely restricted by the lack of functional human cell systems. However,
the successful derivation of pluripotent human embryonic stem (hES) cell lines in the late 1990s is expected to revolutionize biomedical
research in many areas. Due to their growth capacity and unique developmental potential to differentiate into almost any cell type of the
human body, hES cells have opened novel avenues both in basic and applied research as well as for therapeutic applications. In this review we
describe, from an industrial perspective, the basic science that underlies the hES cell technology and discuss the current and future prospects
for hES cells in novel and improved stem cell based applications for drug discovery, toxicity testing as well as regenerative medicine.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Human embryonic stem cells; Differentiation; Cardiomyocytes; Hepatocytes; Drug discovery; Toxicology; Regenerative medicine
1. Introduction
Pluripotent human embryonic stem (hES) cell lines were
successfully derived from the inner cell mass (ICM) of human
blastocysts in the late 1990s [1]. With their unparalleled
growth capacity and unique developmental potential to differentiate into almost any cell type of the human body, these cells
are expected to revolutionize biomedical research worldwide.
The hES cell technology has the potential to open novel
avenues both in basic and applied research as well as in
therapeutic applications.
In many biomedical disciplines, such as drug discovery
and toxicology studies, the lack of functional human cell systems makes the research process inefficient and the outcome
sometimes inaccurate. Based on both clinical and financial
grounds, it is thus crucial to develop innovative tools that
increase the speed of drug development in a cost-efficient and
clinically relevant manner. The access to undifferentiated hES
cell lines and derivatives thereof offers great opportunities in a
wide range of applications, spanning from early target identification and validation studies, via cellular screening and lead
optimization, to the use of functional human cells in toxicity
assessment and safety pharmacology as well as in various disease models (Fig. 1). There is also much optimism concerning
the use of selectively differentiated pluripotent hES cells
in cell therapy, initially targeting degenerative diseases. In
essence, many of the shortcomings in the drug development
process today could conceivably be overcome or improved
by exploitation of hES cell technology, optimally promoting
the discovery of new drugs and treatments.
In this feature, we review the basic science that underlies this exciting field by describing the establishment and
maintanence of pluripotent hES cells along with the directed
differentiation of these cells into functional cell types. We
further discuss the current and future prospects for hES cells
in some novel and improved stem cell based applications
for drug discovery, toxicity testing as well as regenerative
medicine. The focus throughout this review is on hES cellderived cardiomyocytes and hepatocytes, since these two cell
types are central in the drug development process. The derivation and application of other cell types are omitted due to
space limitations and are reviewed elsewhere.
2. Pluripotent hES cells
2.1. Establishment of hES cell lines
There are a number of sources of human stem cells
with varying degrees of developmental potency. Multipo-
Fig. 1. Potential use of hES cells in drug discovery. The use of hES cells and their specialized progenies in drug discovery spans from early target identification
and validation studies, via the use of functional human cells in screening and metabolism studies, to the use of various stem cell technologies in toxicity testing. In
addition, an interesting and prodigious future opportunity is to develop drugs that affect endogenous pools of stem cells to repair local defects in the human body.
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C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) 54–80
tent adult stem cells can be derived from the bone marrow
or from specific organs, while different types of multipotent fetal stem cells also can be obtained from umbilical
cord blood or from aborted human fetuses [2]. The method
for the derivation of hES cell lines [1] was initially adapted
from the previously developed methods for mouse and primate ES cells. To establish a new hES cell line from the
ICM of a blastocyst, the expanded blastocyst is first incubated with pronase to digest the surrounding zona pellucida
(ZP). Subsequently the ZP-free blastocyst is treated with
anti-human whole serum antibody and guinea pig complement. This process, which is termed immunosurgery, lyses
the trophectoderm by an antibody/complement reaction. The
isolated ICM is then placed on a layer of mitotically inactivated mouse embryonic fibroblasts (MEF) feeder cells in a
gelatin-coated tissue culture dish. The initial ICM outgrowth
is usually dissected and transferred to new culture dishes after
7–14 days. Successful propagation of the ICM is associated
with the appearance of cells with undifferentiated hES cell
morphology, whereas contaminating cell types such as primitive endoderm and trophectoderm disappear [3]. The culture
medium used for hES cell cultivation is based on Dulbecco’s
modified Eagle’s medium supplemented with 20% Knockout serum replacement or more historically 20% fetal bovine
serum.
To date, hES lines have been derived in a number of
independent laboratories worldwide using the traditional
derivation method [4–6] or alternative approaches in which
immunosurgery is not performed, such as whole embryo culture or partial embryo culture [7,8]. The hES cell lines can be
maintained in culture indefinitely and exhibit a stable developmental potential to differentiate into all the cells of the
human body. Unlike mouse ES cells, hES cells can also give
rise to trophectoderm-like cells in vitro [9]. The procurement
of hES cell lines has been surrounded by ethical and legal
considerations which, in most parts of the world, have led to
the establishment of guidelines and regulations concerning
stem cell research.
2.2. Cultivation of hES cell lines
The proper maintenance and expansion of hES cells is one
of the most important issues in the study of hES cells as this
procedure generates the starting material for all subsequent
steps and therefore determines the baseline quality. In order
to expand hES cell lines in an undifferentiated and pluripotent
state, the hES colonies are traditionally cultivated on a mitotically inactivated MEF feeder layer (Fig. 2a). The feeder layer
provides certain currently unknown factors, which support
undifferentiated growth of hES cells [10]. Unlike in mouse ES
Fig. 2. Morphology of hES cells in different culture systems. (a) hES cell colonies grown on MEF feeder layer. Scale bar = 100 ␮m, (b) hES cell colonies grown
in feeder-free culture on MatrigelTM . Scale bar = 250 ␮m, (c) hES cell colonies grown on human feeders. Scale bar = 100 ␮m, and (d) EBs from hES cells in
suspension culture. Scale bar = 500 ␮m.
C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) 54–80
cell cultivation, where addition of LIF to the culture medium
is sufficient to maintain the cells in an undifferentiated state,
human LIF does not prevent hES cells from differentiating
[1,5].
The most widely used method to maintain normal hES
cells of high quality is propagation using manual microdissection [1,3,5]. In this delicate process, micropipettes or
finely drawn Pasteur pipettes are used to dissect individual hES cell colonies into small pieces and subsequently
transfer them to fresh culture dishes. This procedure, which
requires particularly refined skills, is usually repeated every
4–5 days. The main advantages of the mechanical transfer
method lie in the absence of cell-dissociating enzymes and
the ability to perform a positive selection at every passage by
isolating undifferentiated hES cells from more differentiated
cells. However, this method is laborious and time consuming,
making it very difficult to process many cells simultaneously.
Due to the disadvantages inherent to the traditional culture
protocols for hES cells, numerous attempts have been made to
develop alternatives which do not require feeder layer preparation or manual passaging. A widely used feeder-free culture
system takes advantage of the fact that soluble factors which
are necessary for maintenance of undifferentiated hES cells
are secreted into the culture medium by the feeder cells. The
hES cells therefore can be grown in the absence of feeder
cells on a growth substrate such as MatrigelTM using a MEF
conditioned medium [11–14] (Fig. 2b). Conditioned medium
is usually prepared by overnight incubation with a confluent
MEF feeder layer and by subsequent filtration prior to its
use for hES cell culture. Other reports indicate that culture
additives which activate the canonical Wnt pathway [15],
a combination of growth factors such as LIF, transforming
growth factor-␤1 (TGF-␤1) and basic fibroblast growth factor (bFGF) [16], a combination of noggin and bFGF [17] or
high levels of bFGF alone [18] may be sufficient to sustain
undifferentiated hES cells in the absence of feeders.
The use of enzymes for cell dissociation during passage
is obviously considerably faster and simpler than microdissection. Therefore, different enzymes such as collagenase
IV [14,19], trypsin [20] and dispase [7,21,22] have been
employed for the expansion of hES cells. During passage,
the hES cell colonies are incubated with enzyme until a suspension of the desired cluster size has been achieved. The
suspension is then transferred to new culture dishes. According to several reports, the use of enzymes for hES cell transfer
may increase the risk of introducing genomic aberrations
during propagation in vitro [23–26]. It is still not clear, however, in which way culture conditions and the occurrence of
chromosomal abnormalities relate to each other.
Several different cryopreservation protocols have been
used for long-term storage of hES cells. The most common
method for traditionally cultured hES cells is vitrification of
microdissected colonies in open or closed straws [3,27,28].
Enzymatically passaged hES cells have also been cryopreserved successfully by slow freezing of small clusters in
cryotubes [13,14,29]. In addition, protocols for the cryop-
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reservation of adherent hES cell colonies have been suggested
[30].
The demand for undifferentiated hES cells as well as
their differentiated progenies for research and regenerative
medicine is expected to increase massively. The culture methods for pluripotent hES cells available today do not allow for
the production of large enough cell quantities to meet this
demand. In addition, the protocols for the transformation of
undifferentiated hES cells to specific functionally differentiated cells are rather unefficient, thus large amounts of the
undifferentiated hES cell starting material are required. Considerable effort is now put into the industrial scale-up of hES
cell production. Even though attempts have been made to
partly automate the traditional manual propagation of hES
cells [31], large scale production of hES cells requires new
culture technologies which are more robust, efficient and
certainly more cost-effective.
More efficient feeder systems, such as different types of
human feeders [21] or immortalized mouse feeders [32], have
been developed and tested for replacement of the laborious
and inconsistent preparation of traditional MEF feeders. The
use of enzymes instead of microsurgery for passage is an
essential prerequisite for the cost-effective production of hES
cells [33]. In addition to the above mentioned collagenase IV
and dispase, we have been using the recombinant enzyme
TrypLE select to passage hES cells maintained on highly
supportive human foreskin fibroblast (hFF) feeder layers in
order to robustly increase the production of undifferentiated
hES cells (Fig. 2c). Such an enzymatic culture method fulfills the requirements for future automation using cell culture
robots to allow further industrial scale-up [34].
Mouse ES cells have successfully been cultured in stirred
bioreactors [35] as well as in perfused reactors [36]. Likewise,
bioreactor technology should have great potential for hES cell
expansion. Continuous medium renewal in cultures has been
found to be beneficial for the maintenance of undifferentiated hES cells as well [37] and different types of rotating
microgravity reactors [38] or stirred vessels [39] have been
employed for cultivation of differentiating hES cells. Therefore, the use of more advanced perfused reactors or hollow
fiber systems for the production of undifferentiated hES cells
appears plausible for future development. Scale-up of hES
cell cultures will remain difficult though, until the factors
that regulate hES cell pluripotency and the maintenance of
the undifferentiated state are discovered and fully understood.
2.3. Spontaneous differentiation of hES cells
In contrast to embryonal carcinoma cells [40], undifferentiated hES cells have an enormous potential to spontaneously
differentiate into various cell types in vitro. Little is known
to date about the mechanisms which can trigger the spontaneous differentiation of pluripotent hES cells into a seemingly
chaotic mix of differentiated cell types. Unwanted spontaneous differentiation of hES cells is observed to a varying
degree under all routine culture conditions and still presents
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one of the major obstacles in hES cell cultivation today. The
unique spontaneous differentiation potential of hES cells can
on the other hand be harnessed to recapitulate certain aspects
of early human embryonic development in vitro and to easily
generate a variety of hES cell progenies. Simple spontaneous
differentiation of hES cells can be initiated experimentally
by discontinuing to passage the hES cell colonies or by withdrawal of conditioned medium. More complex spontaneous
differentiation can be obtained by cultivation of the hES cells
as three-dimensional aggregates, so called embryoid bodies
(EBs) [41]. To initiate the formation of EBs, the hES cell
colonies are removed from the supporting feeder layer and
allowed to aggregate into spheres. The spheres are usually
maintained in suspension culture and give rise to differentiated cell phenotypes of all three germ layers, which arise by a
complex pattern of cross-induction (Fig. 2d). Further differentiation can be obtained by subsequently plating the whole
EBs or cells released from the EBs onto specific extracellular
matrix components.
2.4. Characterization and quality control of hES cell
lines
Human ES cells can be maintained in vitro indefinitely.
The cell lines can be banked and serve as a reproducible, welldefined source for the generation of a large variety of human
cells for various in vitro applications as well as for future
therapeutic purposes. As hES cells can change in culture due
to differentiation or due to genomic alterations, however, it
is essential to maintain rigorous quality management and a
high level of quality control. Principles well known today in
the industrial production of other mammalian cells, such as
master cell banks, working cell banks and controlled batches,
must be applied to the establishment, banking and production of hES cell lines as well. A number of typical hES cell
attributes can be used for their characterization and quality
control.
Pluripotent hES cells can be characterized phenotypically
by their morphology and by their marker expression profile.
Undifferentiated hES cells grow as colonies with distinct borders. The hES cells are small, densely packed and exhibit a
typical cell morphology with a high nucleus to cytoplasma
ratio and large nucleoli. The morphology of spontaneously
differentiating hES cell colonies is clearly different and can
take on many various forms. The colonies may appear to lose
their tight border and cells within the colony may either begin
to enlarge, flatten and separate or may pile up and appear thick
and opaque. Characteristic cell surface markers of undifferentiated hES cells are the stage-specific embryonic antigen 3
(SSEA-3) [42] and SSEA-4 [43], the high molecular weight
glycoproteins tumor rejection antigen 1–60 (TRA-1–60) and
TRA-1–81 [44], and the germ cell tumor monoclonal-2
(GCTM-2) antigen [45]. These markers are downregulated
upon differentiation. Unlike mouse ES cells, undifferentiated hES cells do not express SSEA-1 [46]. Undifferentiated
hES cells display alkaline phosphatase- and telomerase activ-
ity [1]. Furthermore, the human POU-domain transcription
factor Octamer-4 (OCT-4) [47], Nanog [48] and Sox2 [49]
are highly expressed in the undifferentiated state and can be
used to monitor the differentiation status of hES cells. For
the purpose of quality controlling hES cell cultures, combinations of markers can be measured on the protein level
using immunocytochemistry [50], fluorescence activated cell
sorting (FACS) [51] or on the gene expression level using
real-time quantitative PCR [52,53].
A much discussed aspect of hES cells is their genetic
stability in culture [54,55]. Therefore cytogenetic evaluation
must be an important element in the quality control of hES
cell lines. Examples of the most commonly used methods
for cytogenetic analysis of hES cells today are karyotyping,
fluorescence in situ hybridization (FISH) and comparative
genomic hybridization (CGH) [56,57]. Karyotypes are prepared for analysis by staining the chromosomes to reveal their
banding pattern and arranging them as an ideogram. Such
karyotypes are used to study gross chromosomal aberrations
and may be used to determine other aspects of a hES cell line’s
genotype, such as sex. FISH uses fluorescent DNA probes
that bind to specific parts of a chromosome with which they
show a high degree of sequence similarity. Depending on the
combination of probes employed, FISH can be used to detect
and localize the presence or absence of several specific DNA
sequences on chromosomes. CGH allows the analysis of copy
number changes (gains or losses) in the DNA content of cells.
The method is based on the hybridization of fluorescentlabeled abnormal and normal DNA to metaphase preparations
followed by quantitative image analysis of regional differences in the fluorescence ratios. Each of these techniques has
its limitations. Traditional G-banding karyotypes are difficult to analyze due to the low resolution banding pattern in
stem cells, FISH does not allow screening of all the chromosomes of the genome for chromosomal changes and CGH
is unable to detect balanced translocations, mosaicism and
ploidy. Therefore the methods are usually used in combination to complement one another. Finer resolution analysis of
genomic stability is possible by single nucleotide polymorphism assays, which can also be applied to generate genomic
fingerprints of hES cell lines [58]. Another technique with
great potential for the molecular cytogenetic characterization
of hES cell lines is spectral karyotyping. The technique uses
multiple probes to simultaneously visualize all chromosome
pairs in different colors [59]. These methods can be used to
identify chromosomal aberrations when other techniques are
not accurate enough.
ES cells have the capacity for extensive self-renewal but
possess the ability to differentiate along multiple cell lineages
at the same time. Long-lasting changes in gene expression
patterns are required during the progression of undifferentiated ES cells towards more differentiated progeny. Such
inheritable cellular gene expression memory can be controlled by epigenetic mechanisms, such as DNA methylation
or histone modification [60]. Methylation and demethylation of regulatory sequences in the genome are known to
C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) 54–80
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have profound effects on cellular fate [61]. Changes in histone modification and DNA methylation may also perturb
X chromosome inactivation in hES cell lines, an important
mechanism for gene dosage compensation to ensure that
female embryos express similar levels of X-linked genes
to males [62]. Epigenetic changes during culture may have
serious implications for the use of hES cells, especially in
regenerative medicine by affecting the differentiation capacity of the cell lines [61]. Increased knowledge of the human
epigenome is hence expected to contribute significantly to
the future capabilities of hES cell line characterization.
The paramount requirement a hES line has to meet is
pluripotency, i.e. the hES cell line must have the potential
to give rise to derivatives of all embryonic germ layers.
Therefore pluripotency testing is an essential part in the
characterization and quality control of hES cell lines. Pluripotency can be tested in vitro by letting hES cells spontaneously
differentiate via an EB step [3,5]. The differentiated material
is then plated into culture dishes and analyzed immunocytochemically for markers specific for the individual embryonic
germ layers. Currently, the golden standard for assaying the
pluripotency of hES cells is performed by in vivo xenotransplantation of undifferentiated hES cells into severe combined
immunodeficiency mice where the xenografted hES cells
give rise to teratomas [1,3,5]. These tumors contain various
types of tissues representing all three embryonic germlayers (Fig. 3), such as striated muscle, cartilage and bone
(mesoderm), gut epithelium (endoderm) and neural rosettes
(ectoderm). The tissues show a varying degree of differentiation and can be evaluated histologically, thus providing
experimental proof of pluripotency.
2.5. Xenofree derivation and cultivation of hES cells
The ultimate potential of hES cells lies in the clinical transplantation of differentiated cells for disorders which arise
from loss-of-function of a single cell type such as diabetes
or Parkinson’s disease. With respect to any future therapeutic
application of hES cell derivatives it is important to eliminate
the risk of contamination with animal pathogens or immunogenic molecules from the mouse feeder cells or from any
animal derived components in the cell culture medium [63].
Therefore any direct or indirect exposure of the hES cell line
to animal material has to be avoided during derivation as well
as propagation in vitro.
One successful approach to avoid exposure of the hES cell
line to animal material is to replace the animal derived components in the traditional culture environment with human
derived substitutes. The traditionally used mouse feeder cells
have been substituted with several different types of feeder
cells derived from human tissues [16,64–66] as well as with
an autogeneic feeder layer derived from the hES cells [67].
However, the basal medium used in the above studies still
contained animal derived proteins. Complete humanization
of the feeder layer as well as the culture medium was recently
reported by Ellerström et al. [68]. By strict use of only human
Fig. 3. Histology of teratoma from hES cells xenografted to SCID mice.
(a) Neural rosettes (ectoderm), (b) Hyaline cartilage (mesoderm), and (c)
Columnar gut epithelium cells with goblet cells (endoderm). All scale
bars = 100 ␮m.
or synthetic components during the derivation and propagation process a new xeno-free hES cell line was established.
Another promising approach to avoid xeno-exposure of
hES cell lines is the use of defined culture environments
that are not based on a feeder cell layer or on a feeder
cell conditioned medium. Defined feeder-free growth surface
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coatings with individual human extracellular matrix components, such as laminin or fibronectin in combination with
non-conditioned serum-free media supplemented with various combinations of human recombinant growth factors,
have been employed for the culture [69–71] as well as the
derivation of hES cell lines [72].
3. Derivation of functional cells from hES cells
The successful establishment of hES cell lines raises a
whole new set of expectations. One big challenge, however,
is the directed and controlled derivation of lineage-restricted
functional cell types from hES cells. To date, most specialized cells that are derived from hES cells are selected and
propagated from an unsorted mix of various spontaneously
differentiated cells. It will thus become very important to
define the culture conditions and find appropriate isolation
methods that will give rise to a high yield of pure populations of differentiated, functional cells intended for future
applications both in vitro and in vivo.
In this section, we focus on the differentiation of hES
cells into hepatocytes and cardiomyocytes as well as their
respective characteristic properties. The discussion regarding
derivation of other cell types from hES cells is omitted and
the interested reader is referred to other excellent reviews on
this subject [73–75].
3.1. Embryogenesis
During embryogenesis the distinct cell lineages are established. At the stage of gastrulation, a fundamental step in
the development of all types of animals, a gut structure is
formed by drawing cells from the exterior to the interior. A
three-layered structure is subsequently formed in this process, with the innermost layer of the gut tube forming the
endoderm, the outmost layer becomes the ectoderm and the
looser tissue between the two forming the mesoderm. These
three structures are the germ layers that are common to all
higher animals. Together they constitute the first representative organization of the adult body with the gut on the inside,
the epidermis on the outside and the loose and hard connective
tissue and muscle in between.
the liver bud begins to grow, the cells are referred to as hepatoblasts [79]. These cells seem to be bipotential, capable
of differentiating into hepatocytes and cholangiocytes (bile
duct cells) [80]. The hepatoblasts associate with endothelial
cells to form capillary-like structures [81]. Many signaling
molecules and transcription factors are needed for the subsequent growth, maturation and polarization of the liver cells.
Although several molecular details have been discovered in
rodents, the exact gene regulation in human liver development
has not yet been fully characterized [82,83].
3.2.2. Stem cell differentiation to hepatic cells
The liver is the largest organ in mammals and it serves a
variety of important functions. Hepatocytes, the most abundant cells of the liver, perform a number of tasks, including
metabolism of most dietary molecules, detoxification of hazardous agents and storage of glycogen. Through the basal
membrane, the hepatocyte conditions the venous blood coming into the liver by the secretion of soluble factors. Through
its apical membrane, the hepatocyte secretes bile into the
canaliculae that join the bile ducts. The majority of the
remaining cells of the liver are Kupffer cells, stellate cells,
cholangiocytes and various endothelial cells. Altogether,
the different cell types are building up the complex threedimensional structure of the human liver.
Stem cell differentiation into hepatocytes is of great interest, since an access to large numbers of these cells would
enable their use in place of whole organ transplantation as
a potential treatment for severe liver diseases. Of specific
interest in this review is the idea that a readily source of hepatocytes also substantially could facilitate the development
of new drug discovery strategies.
Stem cells as promising sources of human hepatocytes in
the future can be roughly classified into adult and embryonic stem cells. The adult stem cells could further be divided
into intrahepatic and extrahepatic stem cells. The intrahepatic
stem cells are referred to as hepatoblasts, hepatic progenitors
or liver stem cells, and are a natural source of mature hepatic
cells [84–86]. The extrahepatic stem cells can be found in
most organs in the human body and some of them have been
shown to have the capacity to differentiate in vitro or in vivo
to the hepatic lineage [87–90]. This review will focus on the
potential of embryonic stem cells to serve as the source for
mature hepatic cell types.
3.2. Hepatocytes
3.2.1. Hepatogenesis
The definitive endoderm, one of the three embryonic lineages, gives rise to the digestive tract and to organs such
as pancreas and liver [76]. The induction of hepatic genes
requires signaling from at least two different mesodermal
tissues; FGFs from the adjacent cardiac mesoderm and bone
morphogenetic proteins (BMP) 2 and 4 from the septum
transversum mesenchyme [77,78]. These interactions with
the endodermal cells are consequently crucial for early liver
budding phase. When the hepatic endoderm is specified and
3.2.3. Differentiation of hES cells to hepatic cells
For more than 20 years, mouse ES cells have served as a
tool for studying the embryonic development. The first report
on directed differentiation of ES cells to hepatocytes in vitro
was published in 2001 when Hamazaki and colleagues successfully studied hepatic maturation in cultures of mouse
ES cells [91]. This report has been followed by numerous
studies on mouse ES cell differentiation to the hepatic lineage. Although different culturing techniques have been used,
including monolayer cultures [92,93], EB formation [94,95]
and co-culturing with liver cells [96], the development of
C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) 54–80
hepatic cells from mouse ES cells was not efficient and was
always seen in heterogeneous cultures that contained many
other cell types. In a recent publication, Gouon-Evans et al.
used a combination of activin A, BMP-4 and bFGF to differentiate mouse ES cells grown on collagen into a high
proportion of cells positive for ␣-fetoprotein and albumin
[97]. The cells derived in that study also have other characteristics indicative of more mature hepatocytes.
Although the field of hES cell research is much younger
than the corresponding mouse field, a limited number of studies have evaluated the differentiation of hES cells to hepatic
cell types. It was early shown that hES cells has the potential to spontaneously differentiate into such cells [41,98], but
soon thereafter some groups reported on more directed differentiation. Rambhatla and co-workers reported in 2003 that
hES cells differentiated with or without EB formation, in the
presence of 5 mM sodium butyrate and 1% DMSO, will form
cells with hepatocyte-like morphology [99]. No differences
in maturation state could be identified if the EB or the direct
differentiation protocol was used, and the resulting cell types
were found to express hepatocyte-associated markers, such as
albumin, ␣-1-antitrypsin and cytokeratin (CK) 8 and 18. The
hepatocyte-like cells derived in this study furthermore accumulated glycogen and showed inducible activity of one of
the many cytochrome P450 (CYP) enzymes, CYP1A2, measured with the ethoxy resorufin O-de-ethylase (EROD) assay.
One year later, Lavon et al. used a reporter gene construct
regulated by the albumin promoter to isolate hepatocyte-like
cells from human EBs [100]. The eGFP-positive cells could
be sorted using FACS and maintained in culture for a few
weeks. The authors used conditioned media from cultures of
primary human hepatocytes to induce differentiation of hES
cells to a hepatic cell type producing albumin, and moreover
identified acidic FGF (aFGF) as a single factor with positive
effect on these processes. However, in these two first studies
only limited data on the characteristics of the hepatocytelike cells were disclosed. In another study, addition of FGF-4
and hepatocyte growth factor (HGF) induced formation of
hepatocyte-like cells on collagen I or Matrigel coated dishes
[101]. This group studied expression of hepatic transcription factors, such as Forkhead Box A2 (Foxa2, also called
HNF-3␤), HNF-1 and GATA-4. The cells were able to produce urea and albumin, and were able to take up indocyanine
green, the latter suggesting functional transporter systems.
Notably, if the hepatocyte-like cells produced in this study
were cultured for 4 days with Phenobarbital, a significant
increase in pentoxyresorufin (PROD) activity was detected
suggesting the expression of functional CYP2B6 enzyme.
The disadvantage with this differentiation system was the
low efficiency, with a yield of only about 2% of the cells
positive for albumin and CK18. In an interesting study by
Baharvand et al., the hepatic-potential of hES cell-derived
EBs in 2D and 3D collagen culture systems were compared
[102]. To induce hepatic differentiation, several factors were
added to the growing cells, including aFGF, HGF, dexamethasone and oncostatin M. Although both systems gave rise
61
to cells with hepatocyte-like morphology and phenotype, the
3D culture appeared to have kinetic advantages. The yield
of albumin and CK18 positive cells was approximately 50%
in both the 2D and 3D culture system. It was unclear from
this study, however, if functional systems important for drug
metabolism and toxicity in hepatocytes, e.g. the phases 1 and
2 enzymes, were expressed in the two cell populations. In
a very recent study, Soto-Gutierrez and colleagues cultured
EBs on poly-amino-urethane coated polytetrafluoroethylene
fabric [96]. The use of bFGF, a deletion variant of HGF,
dexamethasone and 1% DMSO led to differentiation of hES
cells to progenies with hepatocyte-like morphology. These
cells produce albumin and urea, and were able to metabolize ammonia. Importantly, the authors also indicated that
lidocaine was metabolized by the cells. This is the first data
showing drug-metabolizing effects of any hES cell-derived
hepatic cells.
Data from our own lab show the capacity of hES cells to
differentiate into both hepatoblast- and hepatocyte-like cells
in a two-dimensional culture system (Fig. 4). After about
2 weeks in culture using a specific protocol, cells resembling hepatoblasts are developed. After about one additional
week, these cells have been shown to mature further into
cells with clear hepatocyte-like morphology. The yield of
these cells is about 50%. Moreover, these cells are positive for many mature liver markers and express functional
systems and liver transporters like bile salt acid pump and
organic anion transporter proteins (Biochemical Pharmacology, accepted for publication). For future industrial use of
stem cell-derived hepatocytes, the expression of specific biotransforming enzymes in the cells are of outmost importance.
In a directed study we have therefore addressed this issue by
Fig. 4. Hepatocyte-like cells derived from hES cells. Human ES are differentiated in vitro and after about 2 weeks, cells resembling hepatoblasts
are developed. These cells have been shown to mature further into cells with
hepatocyte-like morphology after approximately one additional week. These
cells are positive for many mature liver markers and they express functional
systems like phases 1 and 2 enzymes as well as liver transporters such as
bile salt acid pump and organic anion transporter protein.
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studying expression and activity of the some important phase
2 enzymes. It was shown that the activities of these enzymes
closely resemble those of human hepatocytes [103]. It should
also be noted that the use of a direct differentiation protocol
is a great advantage compared to the involvement of aggregation cultures, since it is likely to be much more amenable
for large-scale production of cells for industrial applications.
It should be made clear that there is no strong evidence
in any of the described papers whether the hepatocyte-like
cells differentiated from hES cells are derived from definitive
endoderm or not. A huge hurdle in the field is the limited availability of reliable markers for definitive endoderm in human
tissue. Another hurdle is that hES cells are very hard to stably
transfect, something that makes the establishment of efficient
reporter lines tricky. A recently published paper by D’Amour
and colleagues brings, however, new light into the definitive
endoderm issue [104]. Most likely this study will attract more
focused efforts to derive hepatocyte-like cells from a starting
material consisting of pure definitive endoderm. In fact, in
a recent study the derivation of hepatocyte-like cells from
definitive endoderm, was reported [105]. In this protocol,
hES cells were induced by activin A, and further treated with
FGF-4 and BMP-2. The resulting cells showed expression of
hepatic genes and the presence of protein-markers in addition
to exhibiting functions similar to adult liver cells. However,
no metabolism, biotransformation or transport of pharmaceutical compounds was reported. In our own laboratories, we
have taken a slightly different approach for exploiting activin
A induction of definitive endoderm and subsequent derivation
of hepatocyte-like cells (G. Brolén and N. Heins, unpublished
results). It will be interesting to ascertain the further differentiation of the definitive endoderm cells towards the hepatic
lineage. If this will lead to creation of more functional hepatic
cells is however still an open question.
Different groups, including our own lab, have used various protocols to derive hepatocyte-like cells and the level
of characterization performed varies substantially. Some key
issues have been studied, but conclusive data are still lacking
for culture technique (direct differentiation versus aggregate
culture), culture media including factor supplementation, and
surface composition (e.g. feeder cells, collagen/Matrigel or
bioartificial fabrics). Another very challenging future aspect
is maturation of cells in bioreactors giving improved possibilities to mimic the cell–cell signaling and physical parameters
that are affecting hepatocytes during human liver development. In conclusion, no study so far has been able to claim
the presence of high quality hepatocytes differentiated from
hES cells that is derived with a protocol that could be used for
industrialized applications. Even more important, it remains
to be further investigated that a population of such hepatic cells is functional enough to be used for broad drug
discovery and toxicology applications. Critical functions in
this respect are metabolic competence, biotransformation
capacity and transportation of exogenous compounds. Taken
together, much more data on hES cell-derived hepatocyte-like
cells are needed before we can state any such cell population
as fully functional. Since the derivation of functional hepatocytes from hES cells with a scalable method will meet a
huge industrial need, we will most likely see many studies
addressing these and related issues in the near future.
3.3. Cardiomyocytes
3.3.1. Cardiogenesis
The earliest events of organogenesis during embryonic
development are the formation of the heart and the initiation
of its functions. Although the knowledge about the molecular mechanisms that govern cardiogenesis in humans is still
in its infancy, experimental animal models have been very
useful for identifying possible key regulators of heart formation. The initial signals that recruit cells to a cardiogenic
fate are part of the process that patterns the early embryo
[106]. In particular, the endoderm appears to have a directive function for cardiogenesis in the developing fetus [107].
The induction of cardiogenesis is characterized by the expression of transcription factors such as members of the GATA
family of transcription factors, Nkx2.5, Mef2C, Tbx5/20 and
Hand1/2 [108–112]. By initiating the complex myocardial
cross regulatory network these factors are believed to be
involved in morphogenic events leading to the formation of
the four chambered heart [113,114]. In addition, there are several signaling pathways and growth factors which have been
implicated in early embryonic heart formation. Among the
most studied are Wnts/Nodal, BMPs and FGFs [115–118].
3.3.2. ES cell differentiation to cardiac myocytes
In ES cell differentiation cultures, the development of the
cardiac lineage is easily detected among other differentiated
cell types using light microscopy by the appearance of spontaneously contracting areas of cells. It has now been more than
20 years since the first study reported on the capacity of mouse
ES cells to form EBs in suspension with subsequent development of myocardium [119]. Further studies using mouse ES
cells in the early 1990s indicated the usefulness of these cells
for recapitulating cardiogenesis and the development of the
cardiac contractile apparatus in vitro [120–123]. In addition
to mouse ES cells, studies on P19 mouse embryonal carcinoma cells have provided opportunities for in vitro modeling
of cardiomyocyte differentiation [124,125]. Although many
factors and pathways controlling cardiac differentiation have
been identified in the mouse and other model organisms (e.g.
chick, xenopus and drosophila), few have been successfully
verified in the human cell system. The apparent lack of overlap may reflect general differences between the experimental
systems as well as fundamental intrinsic differences between
the species. Notably, the kinetics of the differentiation of ES
cells to cardiomyocytes is different between the murine and
human models. In the mouse system, spontaneously beating clusters of cells appear 1 day after plating of EBs and
within 10 days the vast majority of the EBs contain beating
foci [123]. In the human cells, spontaneous beating generally commences 4 days after EB plating and new areas can
C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) 54–80
appear up to about 30 days post-plating [126–128]. This is,
however, not unexpected since the initiation of differentiation by myocardial and endocardial precursors which leads
up to the formation of the cardiac valves normally takes 12
days in a mouse embryo compared to 35 days in the human
analogue [129]. In any case, these observations underscore
the importance of performing studies using human cells in
order to further our understanding of the molecular events
controlling cardiac development in humans.
Traditionally, the most common way to obtain spontaneously contracting cardiomyocytes from hES cells has been
to differentiate the cells via EBs (Fig. 5) [41,127]. However, the efficiency of cardiogenesis in hES cells has been
reported to vary and 5–70% of the EBs give rise to contracting
cardiomyocytes. This broad frequency distribution probably
reflects variations of the culture conditions used and inherent differences between hES cell lines. Investigators have
worked intensively over the last years to develop more efficient systems for converting ES cells into cardiomyocytes
relying on lessons from studies of the developing embryo. In
mouse and avian embryos it has been reported that primitive
streak and visceral endoderm are important for the processes
that direct cardiac progenitors towards terminal differentiation [130–132]. In an attempt to mimic this situation in vitro,
63
a co-culture system was developed in which hES cells were
cultured together with mitotically inactivated END-2 cells (a
visceral endoderm cell line) in order to support cardiac differentiation [133,134]. However, the END-2 factor(s) promoting
cardiac differentiation of hES cells are still unknown.
Besides the BMP-, Wnt- and FGF signaling pathways,
additional components, such as ascorbic acid, nitric oxide,
Cripto, SPARC, cardiogenol and S100A4, have been shown
to promote or improve cardiomyocyte differentiation in ES
cells cultures [135–140]. Whether these factors have direct
effects on cardiogenesis or if they stimulate certain other
cell populations which in turn activate or inhibit cardiac
development remains to be determined. DNA methylation
also appears to be of importance during cardiomyocyte
differentiation of hES cells and the demethylating agent 5aza-deoxycytidin has been reported to stimulate formation of
beating cells in differentiating human EBs [141,142]. However, this effect was critically depending on the concentration
and timing of administration. In line with this observation,
Noggin (a BMP antagonist) was reported to induce cardiomyocyte differentiation of mouse ES cells in a restricted and
time dependent fashion [143]. Taken together, it is likely that
parameters such as dose, timing, isoform and combination of
factors will require certain attention.
Fig. 5. Morphological illustration of hES cells differentiating to cardiomyocytes. Undifferentiated hES cells were maintained in serum containing medium for 6
days in suspension cultures to form EBs. The EBs were subsequently plated in gelatin-coated cell culture dishes leading to attachment and further differentiation
of the cells. Panel A shows an EB 1 day after plating in a culture dish. Panel B shows spontaneously beating areas present in the outgrowth of an EB 6 days
after plating (dashed circles). Panel C shows a mechanically isolated beating area sub-cultured for 3 days after isolation in a new culture dish. Panel D shows
isolated and enzymatically dissociated single cardiomyocytes derived from hES cells. Spontaneously contracting cells are indicated (arrows).
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To investigate the molecular program involved in early
cardiogenesis, the global gene expression profile of hES
cells differentiating into cardiomyocytes was recently analyzed [144]. This study employed the co-culture system of
END-2 cells cultured together with hES cells in serum-free
conditions [145] and the investigators harvested differentiating cells at discrete time points. Subsequent cluster analysis
identified genes and signaling pathways important for cardiogenesis (e.g. Notch- and Wnt signaling). Furthermore,
several transcription factors, such as Mef2C, Tbx2 and Tbx5,
known to be involved in cardiac development, were upregulated during differentiation and this paralleled the expression
of other known cardiac genes. In addition, this study began
to identify novel genes that could potentially be important
regulators during cardiac lineage specification. Two such
examples are SRD5A2L2 and SYNPO2L, and whole mount
in situ hybridization on mouse embryos showed cardiac
restricted expression of the mouse orthologs of these genes
[144]. Although the expression patterns of these genes indicate an association with heart formation, additional studies
are necessary to confirm their functional contribution to this
process.
In addition to key signaling and transcriptional regulation
of the cardiomyogenic program, recent observations have
suggested a role for muscle specific microRNAs that are
involved in the differentiation of cardiac precursors in the
embryo [146,147]. Potentially, modulation of the levels of
specific microRNAs could be a novel approach for initiating
or sustaining cardiogenesis. An alternative approach could
be to overexpress transcriptional regulators that affect the
differentiation process and direct the cells into the cardiac
lineage. In this regard, overexpression of GATA-4 in P19
embryonal carcinoma cells led to accelerated cardiogenesis
and an increased number of spontaneously contracting cells
following initiation of differentiation [148]. It remains to be
determined whether these approaches will prove useful also
for hES cells.
One of the major obstacles for the utilization of hES cellderived cardiomyocytes (hES-CM) is the insufficient number
of cells achieved by the currently described differentiation
protocols. Further studies will hopefully determine additional
mechanisms and factors involved in cardiogenesis and help
to define the regulatory network involved in the formation of
the heart. The delineation of this complex process will allow
rational design of novel approaches in which the differentiation of hES cells to cardiomyocytes can be more efficiently
directed.
3.3.3. Characteristics of hES-CM
During recent years, cardiomyocytes derived from hES
cells have been characterized on the molecular, structural and
functional level. Although substantial heterogeneity has been
described, in general 30–60% of the cells in isolated beating areas display markers and features of cardiac myocytes
[127,149,150]. The histological appearance of hES-CM is
similar to their mouse counterparts and cells with different
morphologies are observed including round, small, elongated, branched and triangular cells with one or two oval
nuclei and prominent nucleoli [151,152]. In contrast to the
mouse ES-CM, the human analogues only display multinucleation at a very limited frequency (<1%) [150,153]. This
should be compared with the fraction of bi- or multinucleated
adult human cardiomyocytes that is about 20% [154]. Mature
cardiomyocytes also have a more defined rod shape compared
to what is usually observed in cultures of hES-CM where
mixtures of individual cells display different morphologies
[127,141,155].
On an ultrastructural level, transmission electron
microscopy indicated that the hES-CM in early stage contracting areas in general had more disorganized myofibrillar
stacks compared to cells isolated from later stage differentiated cells. Notably, the hES-CM contained Z-band,
gap-junctions and granules of ANP [127,142,155]. Although
several studies have demonstrated the expression of structural elements in hES-CM, the myofibrillar and sarcomeric
organization indicate an immature phenotype. Complicating
the interpretation and comparison of the data across studies
is the fact that most investigators have analyzed the hES-CM
at different stages of differentiation. In an attempt to address
this issue a recent report investigated the in vitro maturation
of hES-CM using electron microscopy [150]. By defining
various stages of differentiated hES-CM from early (10–20
days), via intermediate (20–50 days) up to late stage (>50
days) cells the authors could describe a progressive organization of the sarcomeric pattern. Z-bands were observed
starting from the intermediate phase and at the later stage
also discrete A and I bands were identified in the sarcomeres.
Although the level of maturity of adult cardiomyocytes is not
achieved, these results suggest the possibility to modulate
maturation in vitro using various stimuli (e.g. mechanical or
chemical).
Using standard laboratory techniques, several studies have
documented that hES-CM express a number of important
cardiac markers (Fig. 6). Expression of transcription factors involved in cardiogenesis such as GATA-4, Nkx2.5,
Isl-1 and Mef2c is typically observed also in the hES-CM
[127,141,142,145,155,156]. In addition, structural proteins
including ␣-MHC, ␤-MHC, cTnT, cTnI, MLC-2A, MLC2V, ␣-actinin, desmin and tropomyosin are also expressed
[126,133,141]. Notably, the staining patterns of the structural elements range from cytoplasmic clumps to parallel
bundles of fibrillar structures in different cells, most likely
reflecting the variation of maturation of the hES-CM. Creatine kinase-MB and myoglobin expression suggests that the
cells have metabolic activity indicative for myocytes [141].
Additional cardiac markers such as ANP, connexin 43 and
connexin 45 have also been detected by several independent
groups [141,149,156].
In vitro functionality of hES-CM has been examined
using different pharmacological and electrophysiological
approaches. Cardiomyocytes derived from hES cells typically have a beating rate of about 25–130 beats/min and
C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) 54–80
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Fig. 6. Immunohistochemical analysis of hES cell-derived cardiomyocytes. Human ES cells were differentiated in vitro into spontaneously contracting cardiomyocytes. Contracting areas were mechanically isolated, enzymatically dissociated into single cell suspensions and re-seeded in new culture dishes.
Immuno-labeling of the cells indicates expression of (A) Nkx2.5, (B) GATA-4, (C) cTnI and (D) ANP. The nuclei were counter stained using DAPI (blue).
they can be maintained in vitro for several weeks, sometimes even months, without loosing functionality. In order to
obtain positive or negative chronotropic responses promptly
following drug application the cells need to express specific surface membrane receptors coupled to a signaling
pathway that activate ion channels, membrane transporters
and myofilament proteins. Cardiac pacing is accelerated
through adrenergic stimulation [157] and several studies have
demonstrated that functional ␣- and ␤-adrenergic receptors
as well as muscarinic receptors are expressed in hES-CM
[127,141,155,158].
Using intracellular electrophysiological measurements,
nodal-like, embryonic atrial- and ventricular-like action
potentials have been identified in hES-CM [126,133,141].
In addition to the initiation of action potentials in hES-CM,
voltage-gated Na+ channels are essential for efficient conduction of action potentials through the functional syncytium.
Consistent with the functional significance of Na+ channels in
hES-CM, NaV 1.5 gene expression was detected by RT-PCR
[159] and activities of voltage-gated Na+ and K+ channels
were observed using whole-cell patch clamp analysis [142].
As a complement to patch clamp analysis, modulation of
the electrophysiological properties by ␤-adrenergic and muscarinic receptors in hES-CM can be studied using a micro
electrode array (MEA)-system, which allows non-invasive
measurements of frequency modulation as well as ion channel regulation [127,128,149]. Interestingly, the electrogram
recorded from hES-CM differentiated for 45–60 days was
observed to be similar to the ones recorded from cultured
neonatal rodent ventricular myocytes [160]. Electrophysiological maturation was observed and the QRS amplitude,
QRS dV/dt and conduction velocity increased between day
1 and 3 after plating of the beating cells on the MEA.
Negative force frequency relations and investigations of the
Ca2+ handling machinery using specific inhibitors suggested
that the hES-CM contractions depend only on extracellular Ca2+ and not on release of Ca2+ from the sarcoplasmic
reticulum [160]. The dysfunctional sarcoplasmic reticulum
may be explained by the lack of expression of phospholamban and calsequestrin in hES-CM [161]. Interestingly,
in vitro differentiated hES-CM engrafted and formed an
electrical syncytium with quiescent neonatal rat ventricular myocytes and the excitation-contracting coupling was
blocked chemically by 2,3-butanedione monoxime and heptanol or mechanically by physically separating the hES-CM
from the rat myocytes [158].
Taken together, these results suggest that in vitro
developed hES-CM are functional in that they respond appropriately to a number of pharmacological agents as well as
display basic electrophysiological features. However, the
hES-CM have an embryonic phenotype and they appear to
have an immature sarcoplasmic reticulum function. Additional research is needed to find novel approaches to mature
hES-CM in vitro.
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3.3.4. Cell selection and enrichment strategies
The development of efficient directed differentiation systems is central for increasing the yield of hES-CM. It is,
however, unlikely that the purity of these cell preparations
will be sufficient, thus requiring development of techniques
for separating the target cell population from non-relevant
contaminating cell types. Furthermore, specific cardiac subtypes can be selected using analogous techniques.
Discontinuous Percoll gradients were recently used to
enrich cardiomyocytes obtained from heterogeneous populations of differentiated hES cells [141]. In this study, the
authors reported enrichment from about 20% cardiomyocytes
in the starting preparations to about 70% cardiomyocytes in
the final fractions.
Genetic selection strategies have proven effective for
mouse ES cells in which cardiomyocyte specific promoters
were coupled to reporter genes or antibiotic resistance genes
[162–166]. Cardiomyocytes are selected from the mixed
populations by FACS or selective drug resistance yielding
purified populations. For currently unknown reasons, undifferentiated hES cells have been very difficult to transfect
in order to introduce ectopic genes, but there are encouraging examples of successful genetic manipulation of hES
cells (reviewed in [167]). Importantly, it has been reported
that introduction of constituently expressed reporter genes
in undifferentiated hES cells does not alter the phenotype or
functionality of the cardiomyocytes derived from the transfected cell lines [158,167]. These results lend support to
the further development of specifically targeted genetically
modified hES cell lines designed for studies of the cardiomyogenic program and cell selection. Especially, the role of lethal
mutations could be studied, which is not possible in transgenic animal models.
Mitotic stimulation could be another strategy to obtain
higher numbers of hES-CM. This approach appears not to
be possible in the embryonic, fetal or neonatal mouse cardiomyocytes, since these cells have very limited capacity
to proliferate in vitro [153] and in vivo following engraftment to animal models [168–170]. On the contrary, the
hES-CM have high mitotic rates [141,150,155,171], which
could be exploited to expand the hES-CM populations. However, hES-CM progressively withdraw from the cell cycle
during maturation in culture and thus efforts to increase their
proliferation should be focused at the early stages of differentiation [141,150]. Substantial proliferation of hES-CM has
also been observed in vivo after transplantation of the cells
to nude rat hearts [172]. Reportedly, the xenograft increased
sevenfold in size over a 4-week-time period. In vivo BrdUlabeling and Ki-67 immunostaining showed that up to 25%
of the grafted cardiac cells were proliferating 1 week after
transplantation and this fraction decreased to about 15% 3
weeks later.
3.3.5. Cardiac progenitor cells
There are no particular characteristics that are definitive
for cardiomyocyte precursors, but it is recognized that in
normal course of ontogeny, undifferentiated hES cell first
differentiate into mesodermal cells and then through various precursor stages to functional cardiomyocytes. Primitive
cell populations lacking cardiomyocyte, smooth muscle or
endothelial cell markers have recently been isolated from
mouse, rat, dog and human adult and neonatal myocardium
as well as from cultures of differentiating hES cells
[145,173–176]. These cells, usually referred to as cardiac
stem cells, are characterized by their expression of certain
markers such as c-kit [173], Sca-1 [176], Abcg2 [177,178]
and Islet-1 [174,179]. Theoretically, these populations would
be ideal for up-scaling in vitro since a characteristic feature
of these cells is their capacity to proliferate in culture. Information about the signals that hold committed progenitors in a
proliferative state is lacking, but potentially interesting leads
are mediators of the Notch-signaling [180,181], the GSK-3
inhibitor BIO [182], p38 MAP kinase inhibition [183] and
the PI 3-kinase/Akt pathway [171].
4. Applications of hES cells
The understanding of the genetics and the molecular regulation behind proliferation as well as differentiation of hES
cells has increased in recent years. This will help in developing controlled ways to direct hES cells into specific cell
types, which is a prerequisite before these cells can be used
as tools in drug development or in therapeutic strategies.
There are many ways in which hES cells theoretically
could be used, both in basic research as well as clinically oriented research aimed at developing new drugs and therapies.
Here we highlight some of the current and novel hES cellbased approaches in regenerative medicine, drug discovery
and toxicity testing. The focus is set on cardiomyocytes and
hepatocytes as these two cell types are central in the drug
development process.
4.1. Human ES cells in regenerative medicine
One of the most important potential applications of hES
cells might be the generation of functional cells and tissues for cell-based therapies to repair damaged organs.
Regeneration of failing organs and tissues is limited in the
human body. Some tissues, such as liver, blood and vessels, have an innate ability to regenerate, while many other
vital organs do not. The two major clinically implemented
approaches in current tissue replacement regimes, transplanting organs and implanting artificial tissues, are unfortunately
severely restricted by the limited availability of donors,
immuno-rejection or difficulties in reproducing natural tissues artificially. Measures are therefore urgently needed to
find novel concepts for fully restorative therapeutic intervention. Human ES cells are believed to become imperative in
this new approach to regenerative medicine and could potentially be utilized as a cellular source to replace damaged tissue
by cell transplantation or implantation of cellular scaffolds
C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) 54–80
and as biotools for the discovery of drugs that may activate
resident regeneration-competent cells.
The main argument for the predicted opportunities that
pluripotent hES cells might provide for new strategies in
human regenerative medicine is their potential to generate large numbers of functionally differentiated specialized
human cell types. In addition, hES cells have been found to
be less susceptible to immune rejection than adult cells [184].
These properties make hES cells a unique potential source for
cellular treatment of diverse diseases such as Parkinson’sand Alzheimer’s diseases, type I diabetes and cardiac failure. These disorders affect a large number of people, causing
lower quality of life, premature death and enormous health
care costs. Moreover, despite progress in slowing down disease development or alleviating symptoms, there is still no
long-term improvement or clear-cut cure to degenerative disorders or injury.
Heart related diseases in particular have grown to epidemic proportions in developed countries in recent years. For
instance, ischemic heart disease causes 50% of the mortality
of the population in the Western world and cardiomyocyte
loss related to myocardial infarction is the major cause of
chronic heart failures [185]. Although advances in the prevention and treatment of atherosclerotic heart disease have
reduced the cardiovascular morbidity, the present therapies
rarely results in long-term improvement of cardiac function
[186]. The prognosis of patients diagnosed with heart failure is comparable to patients diagnosed with cancer. In fact,
50% of patients with severe cardiac insufficiency (NYHA
class IV) die within 1 year. The only treatment available is
heart transplantation, but the limited supply of donated hearts
is severely restricting the number of treated patients. The
heart has therefore become an important target in regenerative medicine and will be used here to exemplify the concept
of hES cells in cell therapy. Other cell therapeutic areas are
reviewed elsewhere.
4.1.1. The heart as a regenerative organ
The ability of the human heart to repair damaged tissue
after ischemic injury was previously considered non-existent.
According to this dogma, the mammalian heart is a postmitotic organ without regenerative capacity and with a set
number of cells from birth that gradually is reduced over
time. The only compensating mechanism for loss of heart
tissue is thus hypertrophy and not through proliferation of
individual cardiomyocytes. This view contrasts to the biological reality in newt and zebra fish, where regeneration of
heart tissue is seen after injury, mainly through a mechanism
of blastema formation where de-differentiation of cardiac
myocytes produces proliferative myoblasts [187,188].
However, scientific data published in recent years have
radically changed the notion of human heart muscle cells
as lacking regeneration potential. The dogma has been
challenged by individual researchers [189,190], and the
observation of y chromosome-containing cardiomyocytes in
hearts donated by females to male recipient patients gave
67
new support to the view of the human heart as a regenerating organ [191]. Moreover, each year an estimated 6.4 × 106
cells are lost in a normal heart, indicating that a slow regeneration process keeping the cell number constant or slowly
reduced ought to exist [192]. In several reports a low grade of
cell proliferation has indeed been demonstrated in humans;
14/106 cells undergo mitosis in the normal heart and this is
increased 10 times following cardiac infarction [192,193].
The finding of dividing myocytes in normal and pathologic
heart tissue further supports the hypothesis that there must be
a resident pluripotent stem cell niche in the heart supporting
normal slow regeneration [193]. In recent years, populations
of such cells, having the ability to regenerate myocardium,
have been isolated from mice, rats, dogs as well as humans
[173,174,176,179,194]. These cells could potentially be true
stem cells of the heart. The new data demonstrate that heart
tissue in mammalians has potential of classic regeneration
even though it mainly consists of terminally differentiated
cells [173].
4.1.2. Human ES cells and cell therapy for cardiac
regeneration
Although the human heart has been shown to possess
regenerative capacity, the rate is inadequate to fully repopulate damaged tissue after massive myocardial infarction.
Much optimism is therefore set on cell replacement therapy
for the restoration of injured myocardium.
A number of cell types, for instance mesenchymal stem
cells, skeletal myoblasts and endothelial progenitor cells
[195–197], possess traits that have made them candidates
in clinical stem cell therapy studies. However, none of these
cell populations have proven optimal, with drawbacks such
as lacking important functional properties, requiring ex vivo
expansion or being arrhytmogenic. Human fetal cardiomyocytes obviously have a natural cardiomyocyte phenotype
with appropriate contractile and electrophysiological properties that possibly would make them ultimate donor cells
as far as their functional characteristics are concerned. However, the restricted supply of human fetal cardiomyocytes
together with the ethical issues regarding their use is severely
restraining human therapeutic applications with these cells.
The use of hES cells as a source of cardiomyocytes could
be an attractive option compared to other currently available candidates for a number of reasons: (a) their ability to
differentiate into cardiomyocytes with many phenotypic similarities to adult human cardiomyocytes; (b) their virtually
unlimited growth capacity likely to generate a sufficient number of cells aimed at transplantation; (c) their pluripotency,
making it possible to form also other cell types important
for cardiac repair, such as endothelial progenitor cells and
smooth muscle cells for angiogenesis as well as specialized
subtypes of cardiomyocytes (e.g. pacemaker, atrial and ventricular cells); (d) the cells can be kept in stock and thus
delivered and implanted whenever required; and (e) their
genetic material can be manipulated to promote desired properties.
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To the best of our knowledge, neither hES-CM nor
any other cell type derived from human ES cells, have
yet been used in clinical applications. The functionality of
hES-CM after transplantation, such as engraftment and electromechanical integration, needs to be clearly demonstrated
experimentally in vivo before any clinical usage with these
cells can be considered. There are many reports regarding
transplantation of mouse ES cells into normal or dysfunctional hearts [162,198–207], but in vivo studies with hES
cell are still in its beginnings. There are, however, a few
published research papers to date demonstrating the successful application of hES-CM in animal studies. One of these
studies showed that injected cardiomyocytes generated from
hES cells could engraft and structurally and functionally integrate with host myocardium in immunosuppressed pigs after
transplantation [208]. It further showed that the donor cells
possessed an ability to pace the recipient cardiomyocytes,
i.e. to function as a biological pacemaker, with the depolarization wave originating from the area of implantation. This
study is strengthened by a similar study in guinea pig, which
showed electromechanical coupling of transplanted hES cellderived grafts with host cardiomyocytes, and induction of
rhythmic electrical and contractile activities at the site of cell
transplantation [158]. A third study describes the successful generation of human myocardial tissue by implanting
differentiated cardiac-enriched hES cells into the hearts of
immunodeficient rats [172]. Together these data demonstrate
that hES-CM survive and have the ability to restore myocardial electromechanical properties after transplantation as well
as hold potential for pacemaking applications. Transplanted
ES cells could possibly also have an effect on cardiac repair
by secretion of various factors [209,210] and could therefore theoretically be used as a vehicle for controlled delivery
of therapeutic drugs. These exciting proof-of-principle studies signify the value of hES cells in cardiac regenerative
medicine.
Apart from cardiac dysfunction, there is certainly also
much optimism concerning the use of pluripotent hES cells
as a source for cellular treatment of other diseases associated with degeneration and injury, including Parkinson’s
and Alzheimer’s disease, but this is beyond the scope of this
review.
4.1.3. Concerns with hES cells in cell therapy
Even though cardiomyocytes derived from hES cells are
considered a promising alternative for heart repair in humans,
there are yet safety, ethical and technical concerns that need
to be addressed before the use of these cells can become clinically implemented. Strategies designed to develop refined
protocols for differentiation, isolation, expansion, purification and delivery of hES cell derivatives are crucial to produce
a sufficient number of pure cardiomyocytes or cardioprogenitors that home to the right location. These measures are
also needed to avoid potential major concerns such as differentiation of hES cells into cardiomyocytes without correct
electrophysiological/contractile properties or non wanted cell
types, teratoma formation and arrhythmias. As of today, no
protocol that yields a pure progeny population has been
demonstrated.
The general assumption that hES cell-derived somatic
cells are identical to the corresponding adult cell type derived
through normal development remains to be scientifically
proven. In fact, no such correlation has yet been published
suggesting that caution should be raised when considering
hES cell derivatives for therapeutic interventions. Utilization of cardiomyocytes derived from hES cells, being a type
of non-autologous cells, will also require actions to avoid
problems with immunologic incompatibility which otherwise
would pose a risk of rejection. This approach may include
immunosuppressants, creation of a stem cell bank with different cell surface antigens and/or reduction of differences
between donor and recipient by genetic engineering of hES
cells. In addition, these cells should be generated free of
any animal product to avoid introducing potentially harmful viruses in clinical applications. One step in this direction
was, as mentioned, the first hES cell line derived in xeno-free
conditions recently reported [68].
4.1.4. Activation of endogenous stem cells
In current regenerative medicine, the main attention has
been focused on identifying mechanisms that stimulate heart
muscle tissue re-growth by means of cell therapy [211].
An alternative approach to cell therapy would be to search
for pharmacological substances with a potential to influence
the fate of endogenous stem cells in the human body. The
developmental outcome of stem cells is controlled both by
intracellular cues and the milieu surrounding the cells, i.e.
the stem cell niche. By pharmacologically coaxing these cells
to differentiate into specialized adult cells, local defects in
the body could be repaired. This novel pharmacological concept opens the opportunity for drug discovery in a new field
of organ regeneration, in which hES cells are anticipated to
become an important tool.
Low molecular weight compounds are important in drug
development since they are more stable and less expensive
than polypeptide equivalents. In addition, they have proven
effective in the modulation of the development of stem cells.
Various studies describe the use of synthetic and natural
low molecular weight compounds to induce cell differentiation. For instance, small molecules such as DMSO [212],
retinoic acid [141,166,213], ascorbic acid [139], Cardiogenol
C [140], 5-azacytidine and 5-aza-deoxycytidine [141,214]
have been able to affect the fate of diverse types of stem cells.
However, the effect of many modulators is pleiotrophic. Substances with distinct and directed actions on differentiation,
dedifferentiation or transdifferentiation are therefore needed
to make their clinical use for tissue remodeling possible.
Although the natural processes of dedifferentiation and
transdifferentiation remain to be demonstrated in humans,
there are indications that these mechanisms exist in mammals. First of all, there is evidence that suggests stem cell
plasticity, i.e. cells residing in a specific tissue have the
C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) 54–80
capacity to form other cell types of other organs, designating that these cells are not lineage-restricted [215,216].
Secondly, recent in vitro studies imply that terminally differentiated mammalian cells can undergo reprogramming by
dedifferentiation or transdifferentiation through exposure to
small molecules [217–222]. For example, a study by Chen
et al. describes the ability of the small molecule reversine (a
substituted purine analog) to function as a signal for dedifferentiation of C2C12 myotubes back into progenitor cells,
which is followed by their differentiation into osteoblasts
and adipocytes under appropriate conditions [217]. More
work needs to be performed to ascertain exactly what mechanisms are involved, but the authors speculate that the process
may involve a protein kinase to which reversine binds. Even
though no such pharmacological drug today is available for
in vivo modulation of human cells, the report clearly signifies the potential of small molecular compounds to change
the fate of cells. With a controlled and precise effect of such
a molecule on adult cells or stem cells, the body’s own cells
could therefore theoretically be driven into generating various functional cell types to induce regeneration in vivo. This
approach would also evade many problems, such as rejection
of transplants, usually related to stem cells used clinically.
As proof-of-principle, the work by Chen et al. is therefore
of extraordinary interest and may have landmark importance
for regeneration research and clinical medicine.
The successful sequencing of the entire human genome
has revealed an estimated number of new potential drug targets in the range of 5000–10,000. This is in contrast to the
known number of targets for all marketed drugs in the world,
which is as low as 120. In fact, the 100 top selling drugs in
2001 were directed at only 43 host proteins [223]. The US
Federal and Drug Administration each year approximately
approve the seemingly impressive 30 new molecular entities,
but 90% of these represent compounds regulating targets for
which there already exist drugs on the market [224]. There is
hence a major market for the discovery of unique new drugs
and drug targets that regulate stem cells in specific tissues.
The finding of new drug targets has been hindered by the
lack of screening systems based on normal human functional
cells, which also slows down development of new drugs. So
far, these desired cellular human systems have been primarily
substituted by the use of animal cells or immortalized human
cell lines, which are attractive based on availability and low
cost. However, the clinical relevance of the result from such
cell types is low since these cell systems often lack important
functions present in normal human cells. Animal models are
also used as an alternative, but they are usually both complex and costly apart from not having adequate similarities
to human physiology. Specialized cells derived from hES
cells are believed to become important in the replacement
of suboptimal cell types, since hES cell progenies have the
potential to better represent normal human cells. This will
consequently enhance the precision of the cellular screening
systems, which in turn significantly increases efficiency and
reduces late-stage attrition in the drug discovery process.
69
By using undifferentiated or partially differentiated hES
cells in cell based platforms for drug screening, chemical
compounds that induce or stimulate the differentiation of
cells into specialized cell types can be identified. Through this
approach it may ultimately be possible to develop new drugs
that promote regeneration in situ by activating and mobilizing
regeneration-competent cells. This approach to regenerative
medicine might be favorable to cell therapy as drug treatment
would be available without delay at any time and the health
care costs would be considerably lower. It would furthermore
not necessitate transplantation of foreign cells associated with
various complications, such as rejection. The identification of
substances that induce differentiation of hES cells into specialized cells will also have great impact on our understanding
of signaling pathways and regulatory mechanisms underlying cell and organ development. This generated knowledge
will be very useful for basic research as well as for facilitation
of other therapeutic applications with hES cells.
4.2. Human ES cells for use in drug discovery and
toxicity testing
Therapeutic applications of hES cells are still premature
and are probably years or maybe even decades away. However, hES cells have extreme potentials also beyond their use
in regenerative medicine with applications that are furthermore likely to be more immediate. Two such areas in the
drug development process are drug discovery, with screening as one key activity, and toxicity testing. The hES cell
technology is expected to become particularly important for
purposes where the liver and the heart are central since these
organs have substantial influence on the outcome of drug
effects. There are furthermore no large quantities of human
functional heart or liver cells easily available for cell-based
assays, stressing the need for improved research tools. Here
we will therefore place emphasis on the key role of hES cellderived hepatocytes and cardiomyocytes in non-therapeutic
applications in drug development.
4.2.1. Developmental toxicology
Due to the limited availability and quality of normal
human cells or tissues for in vitro applications most in vitro
toxicity testing today is performed using cells from experimental animals. The results obtained in those experiments,
however, are rather difficult to extrapolate between species
and therefore predictions for humans are sometimes questionable. The model organisms differ from humans in many
aspects, such as in size, appearance, longevity, physiology
and performance. Mouse, the most popular model, diverged
from a common ancestor 75–80 million years ago which has
led to critical differences in anatomy, biochemistry and physiology even at the earliest developmental stages [225]. Human
cancer cell lines or human primary cells isolated from tissue biopsies present the only human alternatives available
today. Transformed cell lines can usually easily be expanded
in culture, but show only a minimal degree of functional dif-
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ferentiation. Primary cells isolated from human tissues are
the best option to date with respect to functionality, but the
material is difficult to obtain and the large variability among
donors limits the reproducibility of any assay performed with
these cells. The promise of hES cells for in vitro toxicology
is the indefinite access to identical starting material, i.e. the
undifferentiated hES cell line to derive a variety of human
cells from.
The field of developmental toxicology combines embryology and toxicology to identify and evaluate substances
which perturb normal homeostasis in the developing organism. Pattern formation and physical growth in the embryo are
regulated by highly complex temporal and spatial signaling
interactions. This exchange of information orchestrates the
finely concerted cascade of cell division, cell migration, cell
death and protein production.
Developmental toxicity testing is traditionally performed
in animal experiments using rodents or in embryo culture
experiments. For ethical reasons, it is obvious that in vitro or
in vivo developmental toxicity testing using human embryos
is impossible. Considerable advances towards an in vitro test
have been made using mouse ES cells for an embryonic stem
cell test (EST) [226]. The EST evaluates the inhibition of
growth and differentiation of mouse ES cells to indicate the
embryotoxic potential of chemicals and has been validated by
the European center for the validation of alternative methods
(ECVAM) in an international study [227]. The original test
has been modified by several improvements throughout the
past decade [228].
The EST clearly demonstrates the potential of stem cells
for developmental toxicity testing, but since mouse ES cells
are used the validity of the predictions for humans is subject to discussion and a human alternative would clearly be
beneficial [229]. The potential for a truly human relevant
assay for developmental toxicity lies in hES cells and their
ability to spontaneously differentiate and to develop tissues
from all germ layers in vitro. This allows the recapitulation
of early human development and can be a unique tool for the
detection and in-depth study of substances which interfere
with the complex processes involved in human embryonic
development.
4.2.2. Applications for hepatocytes derived from hES
cells
Liver metabolism and the interplay between hepatocytes
and other organs are important drug targets for metabolic
and dyslipidemic diseases. In addition, unexpected human
metabolism is today one of the major causes of removal
of potential new drugs from pharmaceutical projects. Moreover, liver toxicity and alterations of liver function are the
most frequent occurring reasons for toxicology among drug
molecules. Unfortunately, the complexity and function of the
liver is not mirrored by any cell type available today. A disadvantage with primary human liver cells is that they rapidly
loose functional properties when cultured in vitro and their
use is therefore dependent on repetitive sourcing. Tradition-
ally, these cells have been isolated from cadavers or cancer
resections. However, the supply of these cells is limited and
phenotypes vary widely among the sourced donors. Available hepatic cell lines contain very low levels of metabolizing
enzymes and they have a distribution of other important proteins that is substantially different from the native hepatocyte.
Thus, there is an urgent need for a model system that mimics human liver cells and that is able to predict the effects
of new candidate molecules in the drug discovery process. In
absence of practically useful human cells, animal hepatocytes
or non-human in vivo models are used to study liver effects.
Pluripotent stem cells may serve as an ideal source of functional hepatocytes. However, so far only limited phenotypic
and functional data are available for the hES cell-derived
hepatocyte-like cells and more research is required in order to
determine the state of maturation and usefulness of the cells.
If these characteristics are verified, the possibilities to apply
in vitro derived humanized liver models in early predictive
strategies dramatically increase.
Studies of metabolism and pharmacokinetic properties
have become a key activity in the drug discovery early screening programs. This is mainly driven by the fact that as many
as 40% of new chemical entities were recognized to fail late
in the clinical phases because of pharmacokinetic problems
[230]. A recent study showed that adverse drug reactions,
most of which are pharmacokinetic based, are the fourth
to sixth leading cause of death in hospitalized patients in
the USA [231]. There is therefore a great need for in vitro
tools to predict pharmacokinetics of new compounds early in
drugs discovery to be able to select high quality compounds
that could be developed into drugs that are safe and easy to
administer. Thus, the pharmaceutical companies have made
major investments to screen for metabolic properties early in
the drug discovery process [232]. Although hepatocytes are
established as the gold standard in drug metabolism studies
[233], a major problem is still the poor predictive power of
the in vitro tools available [234].
Drug metabolism is a major determinant for pharmacokinetics of a compound and hence the potential source
of unacceptable pharmacological and toxicological effects.
Of the many human drug metabolizing enzymes, CYPs
have been responsible for the metabolism of most therapeutics whose elimination is facilitated by metabolism.
CYPs are mixed function monooxygenases and the major
enzymes in phase 1 metabolism of xenobiotics. The oxidative metabolism results in, depending on the nature of the
xenobiotic, inactivation and facilitated elimination, activation of pro-drugs or metabolic activation. The major site of
CYP expression is the liver and CYP3A4 is the most abundant CYP isozyme in human adult liver. The enzymes of
greatest importance for drug metabolism belong to the families one to three, responsible for 70–80% of all phase 1
dependent metabolism of clinically used drugs [235,236].
CYP expression and activity present large interindividual
variations due to polymorphisms. Moreover, CYPs can be
induced several-fold or inhibited by specific drugs, result-
C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) 54–80
ing in additional, although transient, variability of metabolic
activity [237]. Other metabolizing enzymes, which also may
be of importance, are conjugating enzymes like UDP glucurunosyltransferases and flavin monooxygenases [238]. The
early metabolism testing in the drug discovery process is
supposed to answer the following questions: how labile is
the compound to metabolic degradation, to what metabolites
are the compound metabolized, which enzyme is responsible for the metabolism, does the compound induce any drug
metabolizing enzymes and does the compound inhibit any
drug metabolizing enzymes. In addition to metabolic issues,
the role of transporters in the hepato-biliary disposition has
been recently recognized [239]. The transporter protein may
be important for the clearance and elimination of the drug
when it passes the liver. Numerous transporters are available
on the sinusoidal (blood) side of the hepatocyte to mediate
uptake of drugs from the blood as well as flux them back
into the blood stream. Hepatic transporters may also play an
important role in the excretion of drugs and their metabolites
from the hepatocyte into bile.
The metabolic machinery in liver cells is not mirrored by
any in vitro model available today. Primary cells rapidly lose
metabolic and transportation properties. Available cell lines
contain very low levels of metabolizing enzymes and have a
distribution of transporter protein over the cell membrane that
does not reflect the hepatocyte. A model system that could
be used to mimic the human hepatocyte to be able to predict
the metabolic properties early in the drug discovery process
it therefore critical. There is a great interest to explore hES
cells as a stable source for metabolically competent human
hepatocytes. A successful generation of such cells would add
an extremely valuable tool for pharmaceutical drug development. So far, only one example of xenobiotics metabolism in
hES cell-derived hepatocyte-like cells has been reported [96].
However, as soon as more directed efforts to improve and
maintain functional systems in these cells have been developed additional data on metabolically competent cells will
be available.
Human hepatotoxic action of new chemical compounds is
very difficult to foresee and toxicity is many times found late
in the drug discovery process, or in the worst case even after
a drug has reached the market. To a great extent this has its
origin in pronounced species differences, since most experiments designed to address this question are still performed
in various animal models. Great efforts have therefore been
spent in order to establish predictive human liver systems that
could be assayed in vitro. The alternatives available today are
human liver cancer cell lines or human primary hepatocytes
isolated from liver biopsies. Transformed cell lines, such as
the HepG2 human hepatoma line, are easily accessible for
hepatotoxicity testing, but these cells lack many important
functional systems. Human primary hepatocytes are currently
the best option with respect to functional differentiation, but
the acquisition can be difficult and the variability in the material obtained from different donor limits the reproducibility
of any assay performed with these cells. Moreover, the toxic
71
effects of xenobiotics are often dependent on their biotransformation into toxic metabolites and, therefore, the presence
and distribution of biotransforming systems in potential test
systems must be systematically evaluated.
Besides the described phase 1 class of liver proteins,
the phase 2 metabolizing enzymes are of particular interest for hepatotoxicity events. These enzymes take advantage
of electrophilic groups intrinsically carried in a structure or introduced by phase 1 metabolism to conjugate
xenobiotics with donor molecules such as glutathione, UDPglucuronic acid or 3 -phosphoadenosine-5 -phosphosulfate.
As one example, glutathione transferases (GSTs) catalyze
the conjugation of xenobiotics with GSH and are a vital part
of the phase 2 detoxifying system, particularly with respect to
reactive intermediates. Cytosolic GSTs detoxify electrophilic
xenobiotics, such as chemical carcinogens, environmental pollutants and anti-tumor agents. They also inactivate
endogenous ␣- and ␤-unsaturated aldehydes, quinones and
epoxides as well as hydroperoxides, formed as secondary
metabolites during oxidative stress [240]. The conjugates
formed by GSTs are exported by multi-drug resistance protein 1 and 2, which are part of the phase 3 biotransformation
system [241,242]. There are 17 human cytosolic GST subunits divided into seven classes designated Alpha, Mu,
Pi, Sigma, Theta, Zeta and Omega, based on amino acid
sequence similarities [243]. Human GSTs are nearly ubiquitously expressed, but the highest levels are found in liver
and testis [244]. GSTA1 is the most abundant subunit in liver,
whereas GSTP is most frequently expressed in lung and brain.
In a recent study in our lab, hES cell-derived hepatocyte-like
cells were found to express a pattern of GST protein similar to
that of human adult liver cells [103]. In addition, GST activity
is detected in these cells at levels comparable to human liver.
These initial data indicate that hES cells have the potential
to mature into hepatocyte-like cells with functional systems
compatible with hepatotoxicity testing.
A concept test was recently established using mouse ES
cells that showed the potential of these cells to differentiate to the hepatic lineage, thereby giving access to valuable
cell material for assessing hepatotoxicity [245]. However,
no such model is yet described and used for hES cells. At
the end, there is a limited supply of primary human hepatocytes and currently animal hepatocytes are primarily used
for assessing the safety of chemicals and candidate drugs.
Clearly, the need for human material is mounting and the
use of hES cell-derived hepatocytes can potentially provide
an unlimited source of functional human hepatocytes, from
the same genetic donor if desired, and thereby improve the
predictability of toxicity tests and reduce the need for animal
experimentation.
The human liver is an organ consisting of many cell types
other than hepatocytes. For example Kuppffer cells, stellate cells and cholangiocytes are adding important pieces to
the complex liver architecture. Therefore, to be able to fully
understand, and thereby accurately predict, positive and negative effects of new pharmaceutical compounds in vitro, more
72
C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) 54–80
complex models must be developed in the future. This further underscores the huge potential for hES cells as a source
for human hepatotoxicity models, since in principal any specialized cell type can be generated from undifferentiated hES
cells. Athough this remains somewhat speculative, there is a
great hope that hES cell research will provide the first opportunities to mimic simple liver tissue, thereby dramatically
improve the chances to better predict human toxicity in vitro.
However, besides hepatocytes and endothelial cells, derivation of other cell types of the liver has not been reported. It
is therefore likely that it will take several years before more
advanced liver-models consisting of many cell types will be
available for in vitro drug-testing.
In conclusion, functional cells such as hepatocytes derived
from hES cells have the potential to combine a high degree
of specific differentiation with an excellent availability for in
vitro testing of potential new drug candidates. This, furthermore, opens up for new principles for human metabolism and
hepatotoxicity testing that at the same time lead to a significant decrease in animal experimentation. It is also likely that
these cells will find a variety of applications in target identification and validation studies, when human primary cells and
currently available cell lines fail in supplying drug discovery
researchers with cells efficiently making the target of choice
accessible.
4.2.3. Applications for cardiomyocytes derived from
hES cells
The cardiomyocyte is one of the key cell types in cardiac
research, but in the area of cardiac diseases the pharmaceutical industry currently lacks human material for preclinical
drug discovery. Human ES cells can be converted into
spontaneously contracting cells with characteristics of cardiomyocytes [127]. In addition, functional analyses have
shown that the cells are electrophysiologically active and
respond to pharmacological stimuli, suggesting that they have
operational ion channels as well as active adrenergic and
muscarinic receptor coupling systems [133,141,155]. In drug
discovery, these cells would be useful for target identification
and for screening to optimize compound structures of specific
inhibitors. In addition, by using specific disease-carrying hES
cell lines or by genetic manipulation of the cells they can provide disease models and may also be useful for determination
of therapeutic endpoints.
Studies of cardiotoxicity are key activities throughout the
drug discovery programs. The number one safety issue in
development of new drugs is prolongation of QT interval, i.e.
prolonged time elapsing from the start of the QRS complex to
the end of the T wave in an electrocardiogram, corresponding
to a longer total duration of electrical activity of the ventricles.
This has been the primary cause of drug withdrawal over the
last 10 years [246]. Of particular note is the fact that QT prolonging drugs belong to diverse therapeutic classes including
both cardiovascular and non-cardiovascular drugs (e.g. antihistamines and neuroleptics). Specifically, cardiotoxicity is
a well known adverse effect of some cytotoxic drugs and
varies from small changes in blood pressure and arrhythmias
to cardiomyopathy [247]. An issue of significant importance,
especially for young oncology patients, is that long-term side
effects and severe morbidity in surviving cancer patients are
results of cardiotoxicity of the cytotoxic drugs [248]. Hence,
there is a substantial need for strategies aimed at identifying the risk of drug-induced QT prolongation during early
preclinical and clinical phases of drug development.
Electrophysiological methods remain the gold standard
for characterization of ion channel properties. The current
in vitro models utilize cell lines heterologously expressing
human cardiac ion channels, cardiac cell cultures, isolated
tissue preparations and perfused hearts. One of the present
problems is the availability of preclinical models in which
a large library of substances can be screened rapidly. In
addition, some of the current models of cardiotoxicity have
significant limitations and are not always accurately predicting the effect of a drug candidate on humans. The limitations
of the current testing systems are reflected by the market withdrawal of a number of non-cardiovascular pharmaceuticals,
some of which were found to cause QT interval prolongation
and torsades de pointes, a fatal form of arrhythmia [246,249].
Thus, improved models with better accuracy are urgently
needed in order to support the cost-efficient discovery of new
and safer drugs.
Development of high-throughput patch clamp instruments
has together with for example cloned human ether-a-gogo-related gene (hERG) expressing HEK293 cells provided
opportunities for drug safety testing [250,251]. However,
cloned ion channels do not have the proper native environment and structural architecture as the functional hERG
channel in vivo [252]. Since the access to human myocytes is
very limited, researchers have been exploiting experimental
animals in order to find relevant model cells that mimic the
human analogues. The ventricular cardiomyocytes isolated
from guinea pig hearts appear to express ion channels similar to the human cells [253]. One advantage with the isolated
myocytes is that it is possible to obtain high-content physiological information related to all participating ion channels
contributing to the cardiac action potential and this is not
achievable in the cloned cells [254]. The combination of hESCM together with automated platforms for high-throughput
electrophysiological recordings will undoubtedly improve
current in vitro models for ion channel drug discovery. Further combination with novel in silico modeling approaches
will provide cost-effective methods for assessing potential
proarrhythmic risk of novel compounds [255].
According to the regulatory authorities, such as the US
FDA and the European Medicines Agency, specific attention should be directed towards testing pharmaceutical drug
safety in vitro and assessing a substance potential for delaying the cardiac repolarization phase. Using a MEA system
it is possible to detect rhythm, route and origin of excitation, repolarization and conduction in heart tissues or cells.
Thus, heart tissue from mouse or chick embryos in combination with MEA are currently used as a tool to evaluate the
C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) 54–80
potential anti- or proarrhythmic effects of drug candidates as
well as their ability to modulate the repolarization [256]. In
a recent study, the effect of d-sotalol on delayed repolarization was demonstrated in hES-CM using the MEA system
[257]. Although the QT prolongation effect of d-sotalol is
well known [258], this study was the first to experimentally show this phenomenon in a hES-CM model. The same
authors previously had studied the in vitro maturation of hESCM, but it was unclear whether there were differentiation
related effects also on the response to d-sotalol in the cell
preparations [128].
Interestingly, experiments with the hERG-specific channel blocker E-4031 resulted in action potential prolongation
both in embryonic atrial- and ventricular-like hES-CM
suggesting that the IKr (KCNH2) contributes to the repolarization in these cells [126]. In addition, both early and delayed
afterdepolarization were observed. These results hold great
promise for the future development of in vitro assays for
drug development. As indicated above, a common side effect
of cardiac and non-cardiac related drug candidates is their
potential to block hERG channels causing action potential
prolongation and ventricular arrhythmias (e.g. torsade de
pointes) [259]. The possibility to use hES-CM to assay these
potentially fatal side effects would substantially increase the
precision of the tests since the human variants can be analyzed
in a close to native environment.
5. Conclusion
The hES cell research field is still a young discipline.
Despite this fact, the field has advanced substantially since
the late 1990s as described above. It is becoming increasingly
clear that results obtained with ES cells from other species
cannot directly be translated into the human counter part, perhaps with the exception of ES cells from other primates. Thus,
major efforts have to be specifically designated into the hES
cell technology. This is important because of the immense
potential impact hES cells might have on the area of human
medicine and biology in the future.
In the present review we have discussed the establishment
and cultivation of undifferentiated hES cells and it is clear
that the field is strongly moving forward. It is now therefore
realistic to believe that the scientific community within short
can generate hES cell lines in a standardized manner for various applications, including clinical use. Major challenges
still remain for scaling up the production to an industrial
level, but this issue is taken seriously by the scientific community and several major focused up-scaling programs are
being launched. In parallel, it is equally important to develop
robust and efficient characterization methods to verify the
quality of the cells being manufactured. Other key areas
are the establishment of efficient protocols to genetically
manipulate hES cells and derivatives, which will become
increasingly important for the application of hES cells in
practical research.
73
The great expectations of hES cells is based on their
pluripotency and growth capability. A key issue is the
restricted and regulated derivation of functional cell types
from hES cells. Today specialized cells that are derived
from hES cells, including hepatocyte- and cardiomyocytelike cells, are primarily isolated and enriched from a variety
of many other spontaneously differentiated cells. It is therefore crucial to elucidate the exact signaling pathways that
are necessary for lineage-specific differentiation. Recently
developed genome- and proteome analyzing technologies are
anticipated to contribute significantly to the understanding
of the signaling pathways that govern the cascade of events
occurring during differentiation of pluripotent hES cells to
functional cells.
As discussed, there is much optimism concerning the use
of functional cells derived from hES cells in drug discovery
and toxicology. This is driven by the increasing need to move
from animal sources of experimentation towards humanized
tests, in particular cell-based high throughput screens with
both good reproducibility and high clinical relevance. The
hES cell technology is also especially important in areas
where the liver and the heart are central since these organs
highly influence the effect of drugs and toxicants. At present
there is no suitable source of human functional heart or liver
cells in sufficient numbers for cell-based assays, underscoring the prerequisite for improved research tools. To facilitate
adoption of hES cell-derived functional cells as tools by the
pharmaceutical, biotech and chemical industries, the cells
need to be carefully validated and scale-up production and
subsequent manufacture control need to be developed, all
with stable and reproducible protocols.
The potential use of hES cells and hES cell derivatives
in regenerative medicine is possibly the main reason why
public awareness is so high in this particular scientific field.
One exciting future application of hES cells is to employ
these cells as platforms for the development of drugs that
can activate and mobilize endogenous stem cell populations residing in various tissues throughout the human body.
Another headline creating future application is to use cellbased therapies to repair damaged organs and/or functions.
It is clear from the above that hES cells is a unique potential
source for cellular treatment of diverse degenerative disorders
or injuries. Such disorders globally affect numerous people causing both personal suffering and an increased load
on medical services. Even though advances in prevention
and treatment have been made, there is still no long-term
solution to these conditions. Human ES cells are expected
to play a key role in these new approaches to regenerative
medicine.
We are in the very early stages of both understanding what
can be accomplished with the great potential of hES cells as
well as putting into practice the new research opportunity
they provide. With sound guidelines and regulations of the
research in addition to the right resources, the field of hES
cells has a true potential to revolutionize human medicine
and the understanding of human biology.
74
C. Améen et al. / Critical Reviews in Oncology/Hematology 65 (2008) 54–80
Reviewers
Gregory P. Sutton, MD, Medical Director, Division of
Gynecologic Oncology, St. Vincent Hospitals and Health
Services, 8301 Harcourt Road, Suite 202, Indianapolis, IN
46260, United States.
Frederic Amant, MD, PhD, Div. of Gynecological Oncology, Dept. of Obstetrics & Gynecology, University Hospitals,
Katholieke Universiteit Leuven, Herestraat 49, Leuven 3000,
Belgium.
Conflicts of interest
Caroline Améen, Raimund Strehl, Petter Björquist, Johan
Hyllner, and Peter Sartipy are employed by Cellartis AB.
Anders Lindahl is a co-founder of Cellartis AB and owns
stocks in the company.
Acknowledgements
The studies from our lab herein described have generously
been supported by Juvenile Diabetes Research Foundation,
Swedish Animal Welfare Association, Vinnova, Swedish
Research Council, National Institutes of Health and the European Union’s 6th framework programme for research and
development.
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Biography
Peter Sartipy (1971) is a Senior Scientist and Project Manager at Cellartis AB. He received his M.Sc. in Chemical
Engineering in 1994 from Chalmers University of Technology (Göteborg, Sweden). He then went on to earn his Ph.D.
in 2000 from the Faculty of Medicine at Göteborg University. After working as a post-doc at the Department of Cell
Biology at The Scripps Research Institute (La Jolla, CA,
USA) he returned to Göteborg and joined Cellartis AB in
2002. His current research is mainly directed at exploring
hES cell differentiation towards cardiomyocytes and development of novel drug discovery applications based on these
cells.