Use and Application of Stem Cells in Toxicology

Julio C. Davila*,1, Gabriela G. Cezar{dagger}, Mark Thiede*, Stephen Strom{ddagger}, Toshio Miki{ddagger} and James Trosko§

* Pfizer, Inc., Pfizer Global Research and Development, St. Louis, Missouri 63167, and {dagger} Groton, Connecticut 06340; {ddagger} 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

ABSTRACT

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 functional characteristics (Theise and Krause, 2002Go). 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., 2002Go, Sato et al., 2003Go).

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, 1981Go; Nagy et al., 1990Go). 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., 2000Go), and the recently identified Nanog (Chambers et al., 2003Go). Human ES cells were isolated in 1998 (Thomson et al., 1998Go) 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., 2000Go; Reubinoff et al., 2000Go; Rambhatla et al., 2003Go; Xu et al., 2002Go).



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FIG. 1. Origin of embryonic and adult stem cells and their potential applications in toxicology.

 
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 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., 1999Go), brain (Clarke et al., 2000Go), liver (Yang et al., 2002Go), skin (Toma et al., 2001Go), adipose (Zuk et al., 2001Go; Zuk et al., 2002Go), skeletal muscle (Jackson, 1999Go), and blood (Zhao et al., 2003Go). Perhaps the best characterized ASCs is the MAPC isolated from bone marrow described by Reyes et al., (2001)Go. 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., 2002Go). 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. However, 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., 2003Go).

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., 1999Go; Rohwedel et al., 2001Go; Vanparys, 2002Go), as suggested by the European Center for Validation of Alternative Methods (ECVAM) committee (Genschow et al., 2000Go, Spielmann et al., 2001Go). Stem-cell 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 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, 1993Go; Cubeddu, 2003Go). 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., 2003Go); 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., 2003Go, Lavon and Bevenisty, 2003Go). 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 {alpha}-cardiac actin, atrial myosin light chain, ventricular myosin light chain, {alpha}-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 development 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)Go 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 µm 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 a1-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., 2002Go, Toma et al., 2002Go). The ability to expand hASCs in vitro provides unlimited potential for producing quantities of cardiomyocyte-progenitors, 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., 2002Go; Schuetz et al., 1993Go) and for studies of the molecular genetics of drug metabolism pathways (Kuehl et al., 2001Go; Lamba et al., 2002Go). 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, 1981Go), bone marrow (Schwartz et al., 2002Go), blood-system stem cells (Alison et al., 2000Go), and embryonic stem cells (Jones et al., 2002Go; Rambhatla et al., 2003Go). 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., 2002Go). 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. Hepatocyte-like 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 CYP2D6 is expressed at 16% of that found in a normal human liver. The observation of mature hepatic functions in PDSC-derived 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.


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TABLE 1 Characteristics of Hepatocyte-like Cells Derived from PDSC

 
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.


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TABLE 2 Stem Cell Characteristics of PDSC

 
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 premature aging and the diseases of aging (Trosko et al., 1998aGo; Trosko, 2003aGo; Trosko, 2003bGo), 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 apoptosis, 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.



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FIG. 2. Gap-junctional intercellular communication mechanisms.

 
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 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-molecular-weight 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., 2000Go). 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, 1996Go). 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., 2000Go)] or because the connexin proteins are modified by expressed oncogenes to render the gap junctions nonfunctional (Trosko et al., 1998bGo).

In summary, the potential for the use of adult stem cells to assist the field of toxicology has great potential to help develop 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., 2001Go, Kehat et al., 2002Go, Mummery et al., 2002Go) and hepatocytes (Choi et al., 2002Go, Yamada et al., 2002Go). Moreover, embryonic stem cells have been validated as a reliable source for in vitro developmental toxicology studies (Genschow et al., 2000Go, Rowhedel et al., 2001Go, Spiellman et al., 2001Go). 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., 1996Go, Scholz et al., 1999Go, Genschow et al., 2000Go). 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., 1999Go).

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, 2003Go). 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., 2001Go, Pfeifer et al., 2002Go). 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., 2000Go, Hidaka et al., 2003Go).

Molecular enrichment and selection greatly increases the purity of differentiated cells. Klug et al. (1996)Go 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)Go. 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., 2003Go). Muller et al. (2000)Go 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., 2002Go) or oligodendrocytes (Billon et al., 2002Go).

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., 2002Go). Constitutive expression of Nurr-1 also enhanced the functional ability of neurons to produce and release dopamine following depolarization (Chung et al., 2002Go). 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., 2003Go).

A vast and expanding plethora of valuable cell types is currently available from mouse and human embryonic stem cells (reviewed in Gorba and Allsopp, 2003Go); nevertheless, genetic manipulation of ES cells recently produced the extraordinary achievement of oocyte derivation (Hubner et al., 2003Go). ES cell clones expressing GFP under the control of a germ line-specific gene gcOct4, were carefully selected for germ 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.

NOTES

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{at}pfizer.com

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