Download REVIEW Use and Application of Stem Cells in Toxicology

TOXICOLOGICAL SCIENCES 79, 214 –223 (2004)
DOI: 10.1093/toxsci/kfh100
Advance Access publication March 10, 2004
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Use and Application of Stem Cells in Toxicology
Julio C. Davila,* , 1 Gabriela G. Cezar,† Mark Thiede,* Stephen Strom,‡ Toshio Miki,‡ and James Trosko§
*Pfizer, Inc., Pfizer Global Research and Development, St. Louis, Missouri 63167, and †Groton, Connecticut 06340; ‡University of Pittsburgh,
Department of Pathology, Pittsburgh, Pennsylvania 15261; and §Michigan State University, Department of Pediatric and Human Development,
East Lansing, Michigan 48824.
Received December 9, 2003; accepted February 15, 2004
In recent years, stem cells have been the subject of increasing
scientific interest because of their utility in numerous biomedical
applications. Stem cells are capable of renewing themselves; that
is, they can be continuously cultured in an undifferentiated state,
giving rise to more specialized cells of the human body such as
heart, liver, bone marrow, blood vessel, pancreatic islet, and nerve
cells. Therefore, stem cells are an important new tool for developing unique, in vitro model systems to test drugs and chemicals and
a potential to predict or anticipate toxicity in humans. The following review provides an overview of the applications of stem cell
technology in the area of toxicology. Specifically, this review addresses core technologies that are emerging in the field and how
they could fulfill critical safety issues such as QT prolongation and
hepatotoxicity, two leading causes of failures in preclinical development of new therapeutic drugs. We report how adult stem cells
derived from various sources, such as human bone marrow and
placenta, can potentially generate suitable models for cardiotoxicity, hepatotoxicity, genotoxicity/epigenetic and reproductive toxicology screens. Additionally, this review addresses the role and
advantages of embryonic stem cells in the aforementioned models
for toxicity and how genetic selection is employed to overcome
major limitations to the implementation of stem cell– based in vitro
models for toxicology.
Introduction
Stem cells are defined by two essential abilities: (1) they are
able to generate identical copies of themselves, or self-renew,
and (2) they give rise to different cell types. Differentiation is
the process whereby cells acquire new morphological and
This article summarizes in part the Stem Cell Symposium presented at the
42nd Annual Meeting of the Society of Toxicology, Salt Lake City, Utah,
March 2003.
1
To whom correspondence should be addressed at Pfizer, Inc., Global
Research and Development, St. Louis Laboratories, 700 Chesterfield Parkway
North, TA1/T114W, St. Louis, MO 63017. Fax: (314) 274-8613. E-mail:
julio.c.davila@pfizer.com.
Toxicological Sciences vol. 79 no. 2 © Society of Toxicology 2004; all rights
reserved.
functional characteristics (Theise and Krause, 2002). In vivo,
differentiation governs the establishment of somatic cell lineages from germ layer precursors during mammalian development. However, the process is also prevalent in tissue repair
and maintenance during postnatal and adult life, such as in
differentiation of bone marrow stem cells into functional erythroid and myeloid blood cells. The ability of stem cells to
exercise both self-renewal and differentiation, or “stemness,” is
currently the focus of investigation by gene expression profiling (Ramalho-Santos et al., 2002, Sato et al., 2003).
Stem cells can be classified into two major categories, according to their developmental status: embryonic and nonembryonic, or adult, stem cells (Fig. 1). Embryonic stem (ES)
cells are pluripotent cells, isolated from the inner cell mass of
the blastocyst-stage mammalian embryo (Martin, 1981; Nagy
et al., 1990). Pluripotent cells are cells capable of giving rise to
most tissues of the organism, including the germ line during
development. In the mouse, these cells have been the most
important instruments for understanding mammalian gene
function by means of genetic manipulation. In addition, ES
cells express several markers that, altogether, may contribute to
“embryonic stemness” such as Oct4, SSEA1 (Niwa et al.,
2000), and the recently identified Nanog (Chambers et al.,
2003). Human ES cells were isolated in 1998 (Thomson et al.,
1998) and were shown to retain the same plasticity as mouse
ES cells, i.e., the ability to differentiate into several somatic or
somatic-like functional cells such as neurons, hepatocytes,
cardiomyocytes, throphoblast cells, and others (Muller et al.,
2000; Reubinoff et al., 2000; Rambhatla et al., 2003; Xu et al.,
2002).
Adult stem cells (ASCs), also known as mesenchymal stem
cells (MSCs) or multipotent adult progenitor cells (MAPCs),
are specialized cells found within many tissues of the body
where they function in tissue homeostasis and repair. Multipotent cells are precursor cells capable of differentiation into
several different cell types but not all cell types in the organism
like pluripotent cells. Multipotent ASCs can be harvested from
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FIG. 1.
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Origin of embryonic and adult stem cells and their potential applications in toxicology.
organs, grown in culture as a homogeneous adherent population of fibroblast-like cells, and induced to differentiate into
multiple cell types. Maintaining ASCs undifferentiated in cultures is dependent upon culture conditions (i.e., serum and cell
density). ASCs have been propagated from bone marrow (Pittenger, et al., 1999), brain (Clarke et al., 2000), liver (Yang et
al., 2002), skin (Toma et al., 2001), adipose (Zuk et al., 2001;
Zuk et al., 2002), skeletal muscle (Jackson, 1999), and blood
(Zhao et al., 2003). Perhaps the best characterized ASCs is the
MAPC isolated from bone marrow described by Reyes et al.,
(2001). These ASCs have a large nucleus to cytoplasm ratio,
express CD13, stage-specific antigen I (SSEA-I), Flk-1, Sca-1,
and Thy-1, and do not express surface markers CD34, CD44,
CD45, c-Kit, and MHC class I and II. These ASCs maintain
their morphology, telomere length (27 kb), and ability to differentiate even after more than 60 doublings. Implantation of a
single genetically tagged marrow-derived ASC into mouse
embryos resulted in the formation of chimeric adult mice,
which stably expressed the tagged gene in many of their tissues
including the atria and ventricles of the heart (Jiang et al.,
2002). In vitro, ASCs have been shown to differentiate into a
wide variety of cell types such as osteoblasts, adipocytes,
chondrocytes, endothelial cells, skeletal myocytes, glia, neurons, and cardiac myocytes. The majority of these adult stem
cells exhibit differentiation into other somatic cell types. How-
ever, there is a crucial conflict in the field as to whether adult
stem cells give rise to differentiated cell types due to true
pluripotency or to cell fusion (Alvarez-Dolado et al., 2003).
The striking potential benefits of stem cells, when applied to
toxicology, result from their unique properties compared to
primary human cells; that is, unlimited proliferation ability,
plasticity to generate other cell types, and a more readily
available sources of human cells. Stem cell-based systems
offer a very promising and innovative alternative for obtaining
large numbers of cells for early efficacy and higher toxicity
screening, allowing scientists to improve the selection of lead
candidates and the reduction of adverse outcomes in later
stages of drug development. For example, hepatocytes and
cardiomyocytes from stem cells in vitro can be routinely used
to screen new chemical entities for hepatotoxicity and cardiotoxicity, two leading causes of failure in preclinical development of new therapeutic drugs. Moreover, mouse embryonic
stem cells can be routinely employed to screen chemical compounds for teratogenic effects (Scholz et al., 1999; Rohwedel
et al., 2001; Vanparys, 2002), as suggested by the European
Center for Validation of Alternative Methods (ECVAM) committee (Genschow et al., 2000, Spielmann et al., 2001). Stemcell technology provides a new tool for better understanding
the mechanisms involved in drug-induced adverse reactions
and to potentially predict and avoid toxicity in humans. The
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present review will briefly describe some potential applications
of adult and embryonic stem cells in cardiotoxicity, hepatotoxicity, genotoxicity/epigenetic and reproductive toxicology.
Use of Adult Stem Cells to Evaluate Cardiotoxicity
In recent years the issue of a drug’s effect on the QT
intervals has been the subject of increasing regulatory review
and discussion. The QT interval is the portion of an ECG that
represents the time from the beginning of ventricular depolarization to the end of ventricular repolarization. Prolongation of
the QT or QTc interval may be associated with a rare but
potentially life-threatening type of ventricular arrhythmia
known as torsade de pointes (Morganroth, 1993; Cubeddu,
2003). Therefore, the detection of potential effects of a drug
candidate on QT interval prolongation is a surrogate marker for
acquired long QT syndrome and torsade de pointes early in
drug discovery and development. This assessment of cardiotoxicity is imperative before that compound is available for
human clinical trials.
Current preclinical models of in vivo and in vitro cardiotoxicity to drug candidates do not accurately predict clinical
outcomes and have some limitations. For example, telemetrized animals, which provide insight on the effects of drug on
heart function, are expensive and have suboptimal sensitivity.
In vitro systems using Purkinje fibers or cloned human ion
channels fall short of accurately predicting the effect of a drug
candidate on human cardiomyocytes. Cultures of human cardiomyocytes are an excellent in vitro model system (Bird et al.,
2003); however, limited availability of tissue from healthy
donors restricts the availability of primary cardiomyocytes for
high throughput safety evaluation. Therefore, the establishment
of novel test systems to screen new chemical entities (NCEs)
and identify potential cardiac safety liability in humans is
highly desirable. Cardiomyocytes derived from human stem
cells provide a new way to screen NCEs for potential cardiotoxicants and QT interval prolongation and offer a tool to
reduce and possibly avoid unexpected or unwanted proarrhythmic drug effects.
Functional cardiomyocytes have been derived from cultures
of human embryonic stem cells (hESC) (He et al., 2003, Lavon
and Bevenisty, 2003). These human embryonic stem cell–
derived cardiomyocytes (hESC-CM) display the expected morphology (i.e., Z bands and intercalated disks) and express
numerous cardiomyocyte proteins including ␣-cardiac actin,
atrial myosin light chain, ventricular myosin light chain, ␣-myosin HC, atrial natriuretic peptide, and cardiac troponin T and
I. They display rhythmic contractions with a longer action
potential duration (APD) characteristic of cardiomyocytes. Together, these results suggest that hESC-CM are a functional
surrogate for primary cardiomyocytes; however, the controversy surrounding the use of hESCs has limited the develop-
ment of these cells as potential sources of cardiomyocyte
progenitors.
Stem cells derived from adult tissue (ASCs), such as bone
marrow, provide a valuable and more readily acceptable source
of cardiomyocyte precursors in vitro. To this end, Makino et al.
(1999) treated cultures of murine marrow-derived ASCs with
5-azacytidine to stimulate cardiomyocyte differentiation. Following 5-azacytidine treatment, murine marrow cells produced
myotubes with a maximal length of 3,000 ␮m and expressed
cardiomyocyte markers such as cardiac actin, myosin light and
heavy chain, cardiomyocyte-specific transcription factors such
as GATA-4, TEF-1, MEF2A,C and D, and a 1-adrenergic receptor subtypes. These ASC-derived cardiomyocyte progressed in culture to cells that were functionally similar to
cardiomyocytes; that is, they produced a sinus node–like action
potential as well as an action potential with peak notch-plateau
characteristic of ventricular cardiomyocytes. While methods to
derive cardiomyocytes from cultured human ASCs (hASCs)
remain to be developed, culture-expanded human ASCs have
been shown to undergo cardiomyogenic differentiation in situ
following implantation in the heart (Shake, et al., 2002, Toma
et al., 2002). The ability to expand hASCs in vitro provides
unlimited potential for producing quantities of cardiomyocyteprogenitors, which could be employed to assess the effects of
NCEs on cardiac ion channels such as hERG, K ⫹, Na ⫹, and
Ca ⫹⫹, and electrical activity, calcium homeostasis, and contractility could be measured. Such capabilities would provide
valuable early mechanistic insight into potential cardiac electrophysiological effects and improve decision-making at the
discovery and preclinical levels.
Production of Hepatocyte-like Cells from Human Placenta to
Evaluate Toxicity
Human liver has been a useful source of cells for basic and
clinical research. Because of their high-level expression of
drug-metabolizing enzymes (both phases 1 and 2), human
hepatocytes have been extremely useful for the investigation of
drug metabolism toxicology (Raucy et al., 2002; Schuetz et al.,
1993) and for studies of the molecular genetics of drug metabolism pathways (Kuehl et al., 2001; Lamba et al., 2002).
However, the use of large numbers of human hepatocytes in
the basic and clinical studies has created a demand for the cells
that far exceeds the procurement capabilities.
There are a number of possible sources for human hepatocytes other than whole human liver. It is possible that fetal liver
or stem cells from other sources could generate hepatocytes.
There are, however, no reports of complete differentiation of
fetal or any other stem cells to adult human hepatocytes. There
are a number of reports of the derivation of hepatocyte-like
cells from nonhepatic sources, including pancreas (Scarpelli
and Rao, 1981), bone marrow (Schwartz et al., 2002), bloodsystem stem cells (Alison et al., 2000), and embryonic stem
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cells (Jones et al., 2002; Rambhatla et al., 2003). In each
report, some selected hepatic functions are detected in the stem
cell-derived “hepatocytes.” With the exception of the hepatic
differentiation of pancreatic progenitor cells in vivo, there are
no claims of full hepatic differentiation of stem cells.
Strom and collaborators have investigated the possibility of
isolating stem cells from placenta. Preliminary studies have
demonstrated that the placenta contains multipotent stem cells
(Miki et al., 2002). Placental-derived stem cells (PDSC) can be
propagated in culture under relatively simple conditions, which
include a DMEM-based media supplemented with EGF and
5–10% fetal bovine serum (FBS). Under more specific culture
conditions, which include the addition of steroids such as
dexamethasone, the PDSC begin to differentiate along a hepatic lineage as determined by the expression of the epithelial
cytokeratins 8 and 18, and the expression of genes characteristic of hepatic differentiation such as albumin (Alb) and alpha
1-antitrypsin (A1AT) (Table 1). The expression of markers of
hepatic differentiation, such as Alb and A1AT, require the
coordinated expression of liver-enriched transcription factors
such as HNF1, HNF4, and the C/EBP family of genes. These
transcription factors are themselves markers of hepatic differentiation and allow the expression of more differentiated liver
functions. While even immature hepatocytes can express Alb
and A1AT, the expression of most of the drug metabolizing
enzymes is restricted to more mature hepatocytes. Hepatocytelike cells derived from placental stem cells express a number of
the cytochrome CYP450 genes (Table 1), suggesting that the
cells differentiate fully along the hepatic lineage. The expression of PxR and CAR, CYP1A1 and CYP1A2, CYP2A6,
CYP2B6, CYP2C8, and CYP2C9, CYP2D6, CYP2E1 and
CYP3A4 has been detected in these cells using gene array or
real time quantitative RT-PCR analyses (manuscript in preparation). CYP1A1 and CYP1A2 are found to be induced with
beta-Naphthoflavone and CYP2C9 and CYP3A4 with rifampicin to levels similar to normal human hepatocytes (4-10X);
however, none of the CYP genes are expressed at levels higher
than 16% of a normal liver. Based on quantitative PCR,
CYP3A4 and CYP1A2 are expressed at 0.1%, and CYP2B6 at
0.4% of a normal human liver (average of 25 liver cases). Like
in fetal liver, CYP2D6 is expressed early in the maturation
process in stem cell derived hepatocyte-like cells, and
TABLE 1
Characteristics of Hepatocyte-like Cells Derived from PDSC
Expression of
Cytokeratin 8 and 18
Alb, A1AT, c-met
HNF-1, HNF-4 (alpha), C/EBP (alpha and beta)
CYP450 genes (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8,
CYP2C9, CYP2D6, CYP2E1, and CYP3A4)
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CYP2D6 is expressed at 16% of that found in a normal human
liver. The observation of mature hepatic functions in PDSCderived hepatocytes suggests that these cells could be useful in
any process where adult human hepatocytes are currently utilized, including toxicology and drug metabolism studies and
even in the cell therapy of liver disease.
Under specific culture conditions, PDSC can be induced to
differentiate into other cell types. As summarized in Table 2,
under specific conditions, PDSC express characteristics of neural stem cells, and even differentiated neural oligodendrocytes
and glial cells. Studies in Strom’s laboratory indicate that on a
matrigel substrate, cells express functions normally associated
with vascular endothelial cells and even form tube-like structures with rudimentary luminal spaces. Of particular interest,
because of the possibility of using PDSC for clinical cell
transplants in patients with Type 1 diabetes, PDSC express the
transcription factors characteristic of pancreatic islet cells and
messenger RNA for both insulin and glucagon. Pluripotent
stem cells such as ES cells express stage-specific embryonic
antigens (SSEA) and the transcription factor Oct4. Expression
of these products seems to be restricted to pluripotent stem
cells, and expression is lost as the cells differentiate. The
expression of SSEA and Oct4 are readily detected in PDSC by
RT-PCR or immunological analysis by flow sorting. These
data suggest that PDSC may be pluripotent and closely related
to the more widely studied ES cells.
In summary, studies in basic and clinical science, ranging
from the investigation of drug metabolism and toxicology to
the transplantation of human hepatocytes to treat clinical liver
disease, require large numbers of viable human hepatocytes.
Stem cells derived from placenta express many of the characteristics of pluripotent (ES) stem cells, such as the expression
of SSEA and Oct4, and display the ability to differentiate into
a variety of cell types in culture. If the pluripotent nature of the
PDSC is proven and culture conditions can be designed which
will produce large numbers of specific cell types such as
hepatocytes, neurons, or pancreatic islet cells from the stem
cell precursors, the placenta could be a rich source of stem cells
and many different adult cell types for basic and clinical
research. Placental tissue is plentiful, and the tissue is normally
discarded following a live birth. At least as important as the
biological properties of PDSC, the use of stem cells derived
from placenta will not encounter the ethical., moral., political,
or religious objections commonly associated with the use of
embryonic stem cells.
Use of Human Adult Stem Cells to Screen for Genotoxic/
Epigenetic Toxicants and Reproductive Toxicology
Understanding the mechanisms by which physical/chemical
mutagenic, cytotoxic, or epigenetic toxicants lead to death,
teratogenesis, carcinogenesis, atherogenesis, cataractogenesis,
reproductive immunotoxicities, and neurotoxicities, as well as
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TABLE 2
Stem Cell Characteristics of PDSC
Neural stem cells give rise to
Neurons, oligodendrocytes, and glial cells
Vascular endothelial cells
Production of insulin- and glucagon-positive cells in culture
Production of hepatocyte-like cells
Expression of stem cell markers, including SSEA and Oct4
premature aging and the diseases of aging (Trosko et al.,
1998a; Trosko, 2003a; Trosko, 2003b), requires knowledge of
the homeostatic regulation of cell proliferation, differentiation,
apoptosis, and senescence of cells. It is important to understand
that, to date, most mechanistic information derived from in
vitro studies have utilized nonstem, proliferating normal, immortalized, or tumorigenic cells. In many cases, extrapolation
of the results of these studies to the in vivo situation has been
less than perfect. In part, the nonconcurrence from these in
vitro studies to the in vivo situation is due to the fact that in
vivo, adult stem cells, which exist in tissues, might be the target
of or have differential sensitivities to the toxicant, whereas in
classic in vitro cultures, normal stem cells were not present.
Therefore, the critical issue here is that in vitro toxicological
studies must include understanding how adult stem cells respond to mutagenic, cytotoxic, or epigenetic toxicants.
First, while most understand the definitions of mutagenesis
and cytotoxicity, not all agree on the meaning of epigenetic
toxicants. To date, the genotoxicity and the reproductive toxicology paradigm have governed much of the toxicological
screening of chemicals, while assessment of the epigenetic
toxicity of chemicals has been limited. Agents that alter the
expression of genes, either at the transcriptional (methylation
of DNA; acetylation of histones), translational (splice variants
of mRNA), or post translational levels (protein modification by
phosphorylation) could cause cells, particularly stem cells, to
lose control over their ability to proliferate, differentiate, apoptase, adaptively respond, or senesce. Alteration of gene
expression in adult stem cells could have major toxicological
consequences, because these cells are needed to provide progenitors to maintain the healthy state of all organs when cells
of that organ are damaged, are lost due to normal wear and tear,
or become diseased. Many chemicals such as thalidomide, a
nonmutagen and noncytotoxicant, can lead to human teratogenesis by an epigenetic mechanism. Phenobarbital, another
nongenotoxicant, can lead to formation of liver tumors in
rodents. DDT, another nonmutagenic toxicant, can act as a
tumor promoter, immune modulator, endocrine disruptor, and
neurotoxicant. Gossyol and phthalates, yet other examples of
nonmutagenic chemicals, can lead to reproductive dysfunction.
By being able to discern whether a potential toxicant can
differentially cause an adult stem cell to mutate, die by apo-
ptosis, or proliferate abnormally by symmetrical cell division,
or differentiate by asymmetrical division while, at the same
time, it induces different responses from the nonstem cells of
that tissue, one has better mechanistic information for risk
assessment extrapolations to in vivo situations. Illustrating this
point, in developing, mature, and aging individuals, adult stem
cells, by symmetric (self-renewal) and asymmetric (differentiation) processes, provide cells for growth, cell replacement,
and wound repair. The microenvironment (“niche”) of the
adult stem cells in each organ is influenced by cell matrix,
cell– cell adhesion, and extra- and intercellular communication.
The net effect of intracellular signaling of the stem cell determines whether the cell stays quiescent, undergoes apoptosis, or
divides asymmetrically or symmetrically (Fig. 2). Upsetting
this niche microenvironment can alter homeostatic regulation
adaptively or maladaptively, the quality and quantity of the
stem cell pool, their progenitor daughter cells, or the terminally
differentiated cells in any tissue.
A second issue to consider with the use of adult stem cells
for toxicological evaluation of chemicals, with the development of molecular biology techniques such as DNA microarray, is that it is imperative to understand the biology of cells in
vitro or tissues in vivo in experimental design and interpretation of results. To compare one cell type in vitro (control) with
treated cells, or to compare control tissues, treated, or diseased
tissues with DNA microarray technology ignores the reality of
in vivo complex interactions of different proportions and differential sensitivities of the three types of cells in vivo. Most
tissues have a very small number of adult stem cells, the bulk
of the cells being transit or progenitor cells with significant
amounts of terminally differentiated cells of that lineage. Each
cell type expresses different gene patterns, and the transit cells
might be at all stages of the cell cycle, having transitory
stress-induced gene patterns and apoptotic genes expressed.
Clonally derived abnormal tissues found in organs, such as
tumors and atherosclerotic plaques, are hypothesized to arise
from single stem cells whose critical genes have been altered
by either mutagenic or stable epigenetic mechanisms. Reduction of the stem cell pool might be responsible for the aging
process of any organ; hence, ultimately the organism. With the
ability to isolate the few stem cells in any tissue and the
availability of modern molecular biological tools such as the
DNA microarray analyses, the goal of toxicology will be to
characterize these stem cells and compare their sensitivities/
resistance to physical or chemical toxicants to those of their
progenitor and differentiated daughters.
Figure 2 depicts a general concept of how the three cell types
within all tissues, namely, the stem cell, the progenitor or
transit cell, and the terminally differentiated cell of that lineage, are “communicating” with each other via either extra-,
intra-, or gap junctional intercellular communication mechanisms. Hormones, growth factor, cytokines, neurotransmitters,
as well as cell matrix and cell adhesion interactions can be
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FIG. 2.
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Gap-junctional intercellular communication mechanisms.
classified as extracellular communication signalers. The many
discovered intracellular communication mechanisms include
all the signal transduction systems, pH, Ca⫹⫹, c-AMP, ceramides, reactive oxygen species, etc. Gap junctional intercellular communication involves ions and small-molecularweight molecules that can traverse between cells through the
20 known connexin-structured connexons. The net effect of all
these forms of communication-triggered intracellular signals
will determine the cellular fates. Each cell type, in response to
these communication signals, can proliferate, differentiate, apoptose, senesce, or adaptively respond if terminally differentiated. However, please note that stem cells do not appear to
communicate via gap junctions. Also, each cell type appears to
have different extracellular matrix interactions, and stem cells
probably exist in an oxygen-poor microenvironment, with its
particular extracellular matrix interaction helping to maintain
its stem cell niche. The ultimate point of this conceptual
viewing of the different cell types in situ is that exposure to a
potential toxicant will, in all likelihood, induce a different
biological response in the three cell types.
Part of the characterization of human stem cells has occurred, in that one phenotype that seems to be characteristic of
the stem cell is the non-expressed connexin gene and the lack
of functional gap-junctional intercellular communication
(GJIC) (Trosko et al., 2000). This does not seem too surprising
since GJIC has been associated with the control of differentiation; induction of GJIC in adult stem cells is associated with
the onset of differentiation in these cells (Lo, 1996). Interestingly, cancer cells that are thought to arise from stem cells do
not have functional gap junctions, either because the connexin
genes are not expressed (as in HeLa cells) (King et al., 2000)]
or because the connexin proteins are modified by expressed
oncogenes to render the gap junctions nonfunctional (Trosko et
al., 1998b).
In summary, the potential for the use of adult stem cells to
assist the field of toxicology has great potential to help develop
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a biologically based risk assessment of toxic chemical exposure to human beings, as well as the other proposed uses.
Use and Genetic Selection of Embryonic Stem Cells for in
vitro Toxicology
Embryonic stem cells are capable of differentiating into
every cell type of the mammalian organism, and therefore have
intrinsically higher plasticity than adult stem cells. Additionally, embryonic stem cells are highly permissive with regards
to genetic manipulation, whereas adult stem cells are somewhat restrictive in their capacity to be modified with exogenous
DNA. Cardiotoxicity, hepatotoxicity, and genotoxicity studies
can be easily implemented in cell types derived from embryonic stem cells at a large scale due to extensively characterized
culture systems and indefinite in vitro replication ability. Differentiation of embryonic stem cells has enabled cellular
screens on large numbers of functional cardiomyocytes (Kehat
et al., 2001, Kehat et al., 2002, Mummery et al., 2002) and
hepatocytes (Choi et al., 2002, Yamada et al., 2002). Moreover, embryonic stem cells have been validated as a reliable
source for in vitro developmental toxicology studies (Genschow et al., 2000, Rowhedel et al., 2001, Spiellman et al.,
2001). The embryonic stem cell test (EST) is an in vitro
embryotoxicity assay that assesses the ability of chemical
compounds to inhibit differentiation of ES cells into cardiomyocytes, as well as other properties (Newall et al., 1996,
Scholz et al., 1999, Genschow et al., 2000). In comparison to
in vivo studies, the embryonic stem cell test is highly accurate
in predicting cellular toxicity, outperforming classical assays
such as fetal limb micromass and postimplantation whole rat
embryos cultures (Scholz et al., 1999).
The major limitation to standardized high-throughput studies in ES-derived and potentially ASC-derived differentiated
cells is the prominent heterogeneity of cell types that arise from
lineage precursors (Lavon and Benvenisty, 2003). This heterogeneity is prevalent in both simultaneous and induced differentiation of ES cells, where growth and transcription factors
direct in vitro differentiation. In order to generate reliable and
robust cellular toxicology assays, it is imperative to obtain a
population of cells that is strictly pure. The cellular plasticity of
embryonic stem cells can be enhanced in vitro with the use of
molecular tools that confer lineage specificity and selection
ability in differentiated cells. This approach is based on transfection of ES cells with transgenes comprised of cell type–
specific promoters and regulatory elements driving the expression of selectable markers that are activated upon
differentiation (Eiges et al., 2001, Pfeifer et al., 2002). Several
selectable markers are currently employed in both murine and
human ES cells differentiated into lineages of all three germ
layers. Molecular markers engineered into transgenes usually
confer in vitro antibiotic resistance, fluorescence activity for
fluorescence-activated cell sorting (FACS), or both properties,
upon stable integration of the transgene into the genome.
Contaminant cell types are therefore eliminated in culture
following antibiotic selection, whereas desired cell types survive selection. Moreover, expression of fluorescent markers,
such as green fluorescent protein (GFP) in target cells, enables
cell sorting and purification by fluorescence-activated cell sorting for downstream applications (Muller et al., 2000, Hidaka et
al., 2003).
Molecular enrichment and selection greatly increases the
purity of differentiated cells. Klug et al. (1996) obtained 99%
pure cardiomyocytes following expression of the neomycin
resistance gene under the control of cardiac ( myosin heavy
chain. Spontaneous, nonselected differentiated cardiomyocytes
made up approximately 1% of cultures. Molecular and functional properties of hepatocytes were detected following differentiation of ES cells genetically engineered with hepatocyte
nuclear factor (HNF)-3( but not in untransfected controls, as
reported by Ishizaka et al. (2002). Moreover, molecular enrichment confers selective specificity of phenotypes even
within the same lineage. Cardiomyocytes generated in vitro
from a common source of ES cells displayed electrophysiological and gene expression properties of either atrial or ventricular myocytes following genetic manipulation (Hidaka et al.,
2003). Muller et al. (2000) isolated a 97% pure population of
ventricular cardiomyocytes following transfection of ES cells
with EGFP under the control of the ventricular-specific myosin
light chain 2-v (MLC 2-v) promoter. In the same approach,
central nervous system cells that evolve from a neurosphere
precursor can be directed into different phenotypes based on
genetic manipulation according to programmed molecular
switches, yielding dopaminergic neurons (Chung et al., 2002)
or oligodendrocytes (Billon et al., 2002).
Overexpression of transcription factors that are critical to
cellular differentiation is another molecular tool that enhances
the purity of target cell types. When Nurr1, an inducer of
midbrain neuronal cells, was overexpressed in combination
with standard growth factor– directed differentiation strategies,
it doubled the percentage of dopaminergic neurons obtained
from mouse ES cells (Kim et al., 2002). Constitutive expression of Nurr-1 also enhanced the functional ability of neurons
to produce and release dopamine following depolarization
(Chung et al., 2002). Overexpression of transcription factors
may prove particularly valuable when seeking ES-derived cells
that are otherwise rare or inefficient following growth factor–
directed differentiation, such as hepatocyte-like cells derived
from human ES cells (Rambhatla et al., 2003).
A vast and expanding plethora of valuable cell types is
currently available from mouse and human embryonic stem
cells (reviewed in Gorba and Allsopp, 2003); nevertheless,
genetic manipulation of ES cells recently produced the extraordinary achievement of oocyte derivation (Hubner et al., 2003).
ES cell clones expressing GFP under the control of a germ
line–specific gene gcOct4, were carefully selected for germ
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cell surface markers and later yielded follicular structures that
secreted estradiol and generated blastocyst-like embryos by
parthenogenesis. The ability to recapitulate oogenesis in vitro,
in addition to somatic cells, may also provide inestimable value
in reproductive toxicology screening of candidate drugs within
the near future.
The data reported here clearly demonstrated that stem cell
technology has the opportunity to contribute to toxicology of
chemical compounds with great impact. Extensive research is
continuing in order to optimize and validate available resources
to establish reproducible in vitro assays that will directly address cardiotoxicity, hepatotoxicity, and reproductive toxicology. Moreover, the use of stem cell– derived in vitro models
will overcome costly and labor-intensive toxicology studies
performed in primary cellular reagents or in vivo animal models. Additionally, these systems will generate robust data on
human cellular physiology based on the availability of cardiomyocytes and hepatocytes from precursors in the human bone
marrow or discarded placentas. In the future, it is expected that
the advent of stem cell technology will possibly yield reagents
for assays on other cell type-specific agressors, such as neurotoxins and skin, respiratory, and renal toxicants.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Gregory Cosma (Bristol Myers Squibb),
Dr. Jeffrey Johnson (University of Wisconsin), and Dr. Clive Svendsen (University of Wisconsin) for their respective contributions to the Stem Cell
Symposium presented at the 42nd Annual Meeting of the Society of Toxicology, Salt Lake City, Utah, March 2003.
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