[Frontiers in Bioscience S5, 118-133, January 1, 2013]

Current status of human pluripotent stem cell based in vitro toxicity tests

Martina Klaric1, Johannes Winkler2, Kinga Vojnits1, Kesavan Meganathan2, Smita Jagtap2, Roberto Ensenat-Waser1, Juergen Hescheler2, Agapios Sachinidis2, Susanne Bremer-Hoffmann1

1Systems toxicology unit, ECVAM, European Centre for the Validation of Alternative Methods, T.P. 580, IHCP, JRC, European Commission, via E. Fermi 2749, 21027 Ispra,VA, Italy, 2Institute of Neurophysiology, University of Cologne, Robert-Koch-Strasse 39, D-50931 Cologne, Germany

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Use of human pluripotent stem cells in toxicity testing
3.1. Cardiotoxicity testing
3.2. Tests for neurotoxicity
3.3. Tests for hepatotoxicity
3.4. Tests for reproductive toxicity
3.5. Tests for developmental toxicity
4. Emerging technologies in toxicity studies
4.1. Toxicogenomics
4.2. Metabonomics
4.3. Toxicoproteomics
5. Perspective
6. References

1. ABSTRACT

The present review assesses the current status of in vitro tests based on human pluripotent stem cell-derived toxicologically relevant target cells. The majority of the evaluated test systems are in the phase of test development. In particular the success rates of differentiation protocols and their reproducibility are varying depending on different culture conditions but also on the assessed marker panel and the functional evaluation of the cells. However, the amount of differentiated cells decreases in relation to their maturation status. No harmonization has been achieved yet about the required maturation status of the cellular models to be used for toxicological applications. Even with an established cellular model, the selection of appropriate readouts is challenging. Some areas of toxicity, such as developmental toxicity, suffer from insufficient knowledge on predictive biomarkers which leads to difficulties in the selection of the most appropriate endpoints. In this heterogeneous context the rapidly increasing knowledge about 'omics' technologies, might lead to an improvement of the current situation and allow the establishment of more predictive human in vitro toxicity tests.

2. INTRODUCTION

The evolution of a predictive in vitro test based on human pluripotent stem cells (PSC) has to follow a sequence of steps until it can be considered for regulatory purposes. This includes several phases such as test development/optimization, (pre)validation and regulatory acceptance (Figure 1). Each of the different segments seeks to fulfil the criteria as required by the OECD guidance document on the "Validation and international acceptance of new or updated test methods for hazard assessment" and will - compiled as an entire dossier of the test method - form the basis for the evaluation of its validity for implementation in the regulatory decision making process (1).

The present review will discuss the current status of human pluripotent stem cell-based in vitro toxicity tests that have recently been published. Since none of the human stem cell based tests have entered into the prevalidation phase so far, the review will focus on test development requirements for stem cell-based in vitro tests which are the precondition for a test method to enter into prevalidation (2). In summary, this early phase of test development aims to describe the purpose as well as the mechanistic basis of the test and should deliver a sufficiently detailed protocol which might allow the transfer of the method from one laboratory to another. The selection of appropriate controls as well as an initial testing with a training set of chemicals are necessary steps to indicate the reproducibility and reliability of the proposed test (discussed in elsewhere in this issue).

When dealing with human embryonic stem cells (hESC), a careful consideration of the biological and toxicological relevance of the test and a review of existing alternatives to human embryonic stem cells is required. Generally, human health endpoints such as developmental toxicity, hepatoxicity, immunotoxiciy and cardiotoxicity (3) known for their variations between species might benefit from the development of new humanized in vitro tests (3). This is of special importance for other toxicological needs, where no satisfying in vitro model is currently available at all (e.g. male germ cell toxicity), where human pluripotent stem cells might significantly improve the present situation.

3. USE OF HUMAN PLURIPOTENT STEM CELLS IN TOXICITY TESTING

3.1. Cardiotoxicity testing

The development of cellular models relevant for the assessment of the cardiotoxic potential of a substance has recently demonstrated some progress due to the stabilization of the differentiation protocols of various hESC lines as well as of hiPSCs (human induced pluripotent stem cells) (4-6). Currently, the maximum reported differentiation rates towards the toxicologically relevant ventricular cells are of 20-30% (6-7). Emerging 3D culture conditions might improve the differentiation efficiency into nodal, atrial and ventricular cell types. New strategies leading to an upscaling of the cell types of particular interest, e.g. via the establishment of cell lines of precursor cells, might be another way forward to overcome the current shortcomings of a specific cell type.

The selection of an appropriate marker panel allowing the monitoring of the maturation status of cardiomyocytes is of high relevance since the degree of maturation of the derived cells might impact their toxicological responses. In the cardiac system, the marker expression differs between embryonic/fetal-like 12-day-old cardiomyocytes (7) and cardiomyocytes with a more adult profile that have been differentiated for 60 days in culture (6). A well-defined marker panel should serve as basis for acceptance criteria supporting/allowing a reduction of intra- and interlaboratory variability of the test system and is a prerequisite for toxicological in vitro tests entering into more formal validation efforts.

In vitro cardiotoxicity tests that are currently under development focus mainly on two adverse health effects: arrhythmias and drug-induced cardiomyopathies. The release of cardiac troponin T and the fatty acid binding protein 3 are clinically decisive cardiac biomarkers for the various forms of cardiomyopathies. In their system, Andersson et al., have monitored the release of those two biomarkers after doxorubicin treatment by using biosensing methods (4). Nevertheless further studies will be necessary to fully prove the toxicological relevance of this system. Severe adverse effects such as Torsade de Pointes (TdP) are associated with chemical interactions with various ion channels of the heart (8). Two test systems analyze the extracellular field potentials of hESC-derived cardiomyocytes and recently a novel test system was developed that uses iPSCs from a patient with long QT syndrome (7, 9-10). One of the most advanced is the test system of Braam et al. that has performed a feasibility study with 12 compounds with well known effects on the different ion channels to demonstrate the biological and toxicological relevance of their test system (7). This and other currently available test systems are summarized in Table 1. Further efforts are now needed to define the intralaboratory variability as well as a robust procedure to interpret the data obtained by testing unknown compounds.

3.2. Tests for neurotoxicity

The development of in vitro tests for neurotoxicity and developmental neurotoxicity has always proven to be challenging. In vivo tests include the data about prenatal and postnatal development and can assess the effects of a chemical on both the morphology and functionality of the nervous system. At the moment, various protocols have been established to differentiate PSCs into neural precursor cells (11), and terminally differentiated cells (motoneurons (12), glia cells (11), dopaminergic neurons (13-14), cerebellar cells (15)).

The protocols based on hESCs usually yield a high percentage of neural precursors (more than 80%) (16) and a significant number of target cells (up to 60%) (17). Similar results can be obtained with iPSCs (16). iPSCs also give an opportunity to create patient- and/or disease-specific models for neurotoxicity testing as was done in the work of Ebert et al. who generated iPSCs from a patient with spinal muscular atrophy and differentiated them towards motoneurons (18).

General RNA and protein markers for neural precursor cells are quite well established and include among others NESTIN, SOX1, PAX6, NEUROD (19). However, the choice of markers for specific lineages of stem cell derived-neuronal cells can vary between the different authors, which makes the results hard to compare. In the case of murine ESCs, an extensive review of the markers for the different lineages was published recently (20) and a similar agreement in the field of human stem cell-derived neuronal cells might prove as useful for the comparison of differentiation protocols.

The major drawbacks of currently available protocols (Table 1) are the low yield of some cell types (19) and the long culture periods for the differentiation (up to two months) (11, 18, 21). However, in some systems it has been possible to produce a large stock of progenitor cells that could be differentiated at a later stage into the desired cell type. Furthermore, pluripotent stem cell-derived neuroprogenitor cells are already commercially available (22-23) and can be differentiated by the end user. This could significantly shorten the time needed to obtain the target cells for toxicological applications.

Two tests have been developed in order to measure the effects of chemicals on dopaminergic neurons (14, 24). Han and co-workers have developed a screening platform for the toxicity testing of compounds on neural progenitors and differentiated neural cells. Their aim was to find compounds that would selectively kill the progenitor cells, leaving a pure population of differentiated cells that would be used for therapeutic purposes (22). This test system could be adapted also for the screening of compounds for potential neurotoxic effects on three stages of neuronal differentiation. The second test was developed by the US EPA. The system is based on the automated high content image analysis of the effects of chemicals on neurite outgrowth that allows for a medium to high throughput screening of chemicals (23).

The future challenge for test developers in the field of neurotoxicity is the establishment of test systems that could predict the effect of a chemical also on the functionality of the neuronal cells (e.g. electrophysiological properties, neurotransmitter activity, etc.). The described test systems are now offering the opportunity to determine the crucial biological pathways that following perturbation might lead to adverse effects in vivo. Highly standardized in vitro models are a prerequisite for a reliable pathway analysis.

3.3. Tests for hepatotoxicity

The lack of metabolic competence turned out to be a major bottleneck for the regulatory acceptance of in vitro toxicity tests (24). Furthermore, it is well known that the liver is one of the major targets of chemically induced adverse effects (25). The crucial characteristics of an in vitro liver model are the presence of a vast array of enzymes and a level of enzymatic activity that should mimic the in vivo situation. Major efforts are being currently undertaken to set up cell-based models allowing to address metabolism-mediated toxicity and hepatotoxicity. Of the currently available in vitro liver cellular systems, primary hepatocytes are problematic due to their rapid dedifferentiation during in vitro culture (26). Other models are mainly based on cells of carcinogenic origin. Malignant cells often show unwanted characteristics and, compared to primary cells, exhibit a lower functionality which could affect the toxicological and biological relevance of the in vitro system (27).

The first reports on hepatic cells derived from human pluripotent stem cells raised hopes to use human pluripotent stem cells as a basis for the generation of metabolically competent hepatocytes (28-30). In the last years several groups have established successful differentiation protocols into hepatocyte-like cells that express relevant markers that are found during liver development in vivo (31-33). Despite this progress, the differentiation protocols still vary in the combinations of growth factors used to drive the differentiation into various stages of hepatocyte development (see Table 1). In particular activin A, fibroblast growth factor 2, oncostatin M and BMP4 seem to be crucial for this process (34-36). Other factors such as LY294004, a phophatidylinositol 3-kinase inhibitor, provide support for further differentiation of cells along the hepatic lineage (37). Chiao et al pointed out signaling pathways that may be manipulated in order to direct more efficiently the differentiation of hESCs towards mature hepatocytes. Those pathways were revealed by the isolation and transcriptional profiling of purified hepatic cells derived from human embryonic stem cells (38).

Various liver-associated genes and transcriptional factors have been proposed for the close monitoring of the successful differentiation and maturation of hepatocyte-like cells when establishing the optimal culture conditions. On the other hand, the functionality of hESC-derived hepatocyte-like cells was proven by their capacity to produce urea and albumin, store glycogen and have functional metabolizing enzymes critical for toxicant biotransformation (39). The majority of published protocols are using the hESC lines H1 and H9 (WiCell Research Institute) but Song et al. demonstrated that also two iPSC lines can differentiate into hepatocyte-like cells to the same extent as H1 hESCs after stimulation with a series of growth factors (40). A comprehensive overview on the differentiation of stem cells into hepatic lineages and related markers was provided by Snykers et al. (41). Despite the advances in the differentiation protocols, a clear definition of the markers and functionalities a pluripotent stem cell-derived hepatocyte should have, to be acceptably used in toxicity testing, has not been achieved so far.

3.4. Tests for reproductive toxicity

The fields of reproductive and developmental toxicity testing are very complex as they include all phases from gamete formation up to birth, including fertilization and prenatal development. In the last years, efforts have been made to develop a series of test systems that use human pluripotent stem cells to detect the effects of chemicals on the reproductive system.

One of the most important targets in reproductive toxicity is the gametogenesis. Despite the fact that murine ESCs have been differentiated into early germinal cells and spermatozoa, the differentiation of human pluripotent stem cells into gametes is still under development. Efforts have been made to stabilize the differentiation of germ cells in vitro, in particular into male gametes (see Table 1). However, the amount of cells that can be obtained with the described protocols is very low (5%) (42-44). Different groups have reported the expression of markers of male primordial germ cells and in some cases even some meiotic markers (44-46). Two different groups have successfully differentiated hESCs into relatively mature haploid spermatids that were showing the first signs of flagellum development and acrosin expression (42-43). The development of protocols for the generation of female gametes has not been successful yet, even if the work done by Aflatoonian and colleagues has shown the increase of female gamete markers after in vitro differentiation (42). Nevertheless it still remains unclear which would be the functional status of such in vitro derived gamete-like cells and whether their functionality would be relevant for toxicity studies.

3.5. Tests for developmental toxicity

Another important chemical target is embryonic development. The test systems for developmental toxicity that are currently being developed are mainly focused on the early phases of cell differentiation and maturation. Since developmental toxicity testing suffers from the fact that many mechanisms of developmental toxicants are unknown and predictive readouts are lacking, more research is required to define new biomarkers and a detailed understanding on the pathways that -if affected- will lead to pathologic findings. Emerging technologies such as "omics" and the automation of in vitro testing are important steps forward in order to define the toxicological information that is needed to make reliable forecast on the hazard of a chemical (47-48). The mechanisms leading to interspecies variations are not well known this is why clear concepts are required on how to validate novel in vitro models based on human cell types.

Stummann and colleagues have developed a test mimicking the early development of the human nervous system and have challenged it with the well-known developmental neurotoxicant methylmercury chloride (MeHg) (49). This system provides a tool to assess the different effects of chemicals specifically during the generation of neural precursors and mature neural cells. Nevertheless the application of this test for developmental toxicity testing would require a bigger number of chemicals to be assessed and also the establishment of a suitable data interpretation procedure. In any case, this test system only elucidates the chemical affects on ectodermal differentiation. Because of the complexity of embryonic development and the possible perturbations by chemicals of all three germ layers other test systems relying on cells originating from the mesodermal and endodermal layers should be established and be further analyzed. Currently it is not fully understood which cell types (or their precursors) exhibit crucial pathways and should get priority for assessing chemical effects. Special attention should be given to the windows of sensitivity during embryogenesis which means that not only terminally differentiated cells are of interest but also the correct temporal pattern of differentiation of their precursors (50-51).

Despite the progress that has been made in the last years, the methods that are currently in the phase of development still need to be refined regarding the amount of target cells as well as the readouts and data interpretation procedures. In a recently published draft report on alternative methods for cosmetics testing it was concluded that several years of test development will be still necessary before a test for developmental toxicity based on human pluripotent stem cells can enter into the validation process (52).

4. EMERGING TECHNOLOGIES IN TOXICITY STUDIES

Currently most of the efforts in stem cell based toxicity testing have been devoted to the standardization of the cellular models which is the one integer part of an in vitro test. However, another crucial parameter in the development of an in vitro test is the correct selection of the readout which reflects the toxicological endpoint one wants to assess with the in vitro test (e.g. the use of multielectrode arrays as readout for assessing Q-T prolongation). That the selection of the readout is not trivial becomes obvious when the exact mode of action of the chemical is not fully understood. In this case sophisticated technologies such as "omics" technologies are necessary to elucidate the chemical's mechanism of action. According to this information the selection of a readout for the in vitro system will be more focused. The experience with the validated murine embryonic stem cell test which relies on the appearance of beating cardiomyocytes after treatment with embryotoxicants (53), has demonstrated the high relevance of selecting predictive readout systems. This readout system must be associated with a detailed understanding of what is happening in the cell system. Good examples for the selection of predictive readouts are transcriptional and binding assays (54-55) that can provide information on the estrogenic and androgenic properties of chemicals. In the previous section we have shown that several toxicity tests based of stem cells are in the phase of development. The readouts differ from test to test and are sometimes difficult to compare. Some of the readouts are not adapted for high throughput screening which is becoming essential in toxicity testing where there is a need to understand the prevalence of a toxic effect in a universe of chemicals or to identify patterns of toxicity across various cellular models. The need for a higher throughput and identification of novel biomarkers of toxicity that would be useful in different test systems has fostered the application of new technologies in in vitro toxicology.

Emerging technologies now drive the establishment of novel readouts and biomarkers that lead to the definition of even more criteria to properly characterize and define the adequate functional ESC/iPSC-derived somatic cells required for toxicity testing purposes. Cell culture techniques should co-evolve with such techniques to apply them from the very beginning and to avoid later adaptations that might delay the test development process. Among the different high throughput (HT) technologies emerging right the "omics" technologies seems to be very promising for toxicity testing.

The recent advances in genomic research significantly contributed to the discovery of new toxicity pathways and the understanding of toxicological mechanisms on cellular and molecular level (reviewed in (56)). The technologies measuring the quantitative changes in molecules such as RNA, proteins and intermediary metabolites on a global level are often termed "-omics" technologies, encompassing toxicogenomics, toxicoproteomics, including high content imaging, and metabonomics. Global analyses of biological responses are based on the concept that the disturbed cellular and molecular functions do not only permit detection of toxicity, but can also help to understand mechanisms and pathways of toxicity. It is hoped that these technologies will explore possible common mechanisms of toxicity and contribute identifying specific biomarkers of toxicity, independent of species or cellular model system used.

4.1. Toxicogenomics

Toxicogenomics measures the modulation of gene expression in response to exposure and explores the basic mechanisms of toxicity. Ideally, changes in gene expression levels correlate to traditional toxicological readouts such as changes in cellular response and organ structure induced by the toxicant. For measuring gene expression, DNA microarrays can be used to measure the differential expression of thousands of genes at the same time. A variety of array platforms such as cDNA arrays, high density oligonucleotide and oligonucleotide bead arrays have been used (57-58). For examining defined sets of genes, custom-made microarrays are also available (59).

The generation of high content microarray data is only the first step for understanding the mechanism of toxicological responses. Correlation of gene expression signatures to toxicological endpoints is essential for prediction of toxicity by expression profiling. Functional classification and assignment of transcripts regulated after exposure to toxicants will allow identification of pathways of toxicity (Toxicity of the 21st century) (59). As an example of this approach transcriptomics data were successfully used to classify toxicants according to their mechanism of action. Rat hepatocyte cultures were treated with 15 known hepatotoxicants. The trancscriptomics analysis of the gene expression profiles of the different toxicants showed that, despite the fact that each toxicant has a specific signature, they could be clustered according to their similar toxicological mechanisms. The clustering of these compounds was concordant with previously published in vivo reports (60).

Huang and co-workers compared cisplatin-induced nephro- and hepatotoxicity in vitro and in vivo using focussed microarrays. The microarray studies succeeded in identifying pathways of cisplatin-induced nephrotoxicity demonstrating discrepancies between in vivo and in vitro findings. Whereas in vivo, the kidney was the primary target organ, hepatic toxicity was more pronounced in vitro (61). Also Jagtab et al. propose a transcriptomic approach in hESCs for monitoring specific toxic effects in early embryonic development (62). Cytosine arabinoside (Ara-C) was used as reference compound and demonstrated a specific induction of neural genes such MAP2, TUBB III, PAX6, TH and NESTIN whereas mesodermal markers (HAND2, PITX2, GATA5, MYL4, TNNT2, COL1A1) were inhibited at day 14 of differentiation.

The relevance of toxicogenomic approaches in safety testing is widely recognized. In a retrospective study of non-clinical safety studies, Foster et al. concluded that significantly regulated transcripts can serve as robust biomarkers of toxicity. While correlation with histopathological data was limited, transcriptional changes often preceded traditional endpoints, demonstrating the sensitivity of transcriptomics. The major challenges of evaluating toxicogenomics data are the integration with traditional histological data, the agreement on data analysis methods and procedures for biomarker identification (63).

4.2. Metabonomics

The term metabonomics refers to the quantitative measurement of the dynamic multi-parametric metabolic response of living systems to pathophysiological stimuli or genetic modification (64).

Metabonomics-based toxicity studies are mainly performed using samples from in vivo studies, including blood plasma, urine, saliva or cells. After preparation, samples are analysed by either nuclear magnetic resonances (NMR) or mass spectroscopy (MS) technologies. The resulting metabolic profiles are then evaluated by a multivariate statistical analysis to identify significant differences in metabolite repertoires and to propose candidate markers, as it was recently suggested (65-66).

In 2007, Cezar and colleagues were the first to analyze hESCs with metabonomics approaches for developmental toxicity testing. Their platform was capable to identify alterations in the metabolic profile of hESCs exposed to valproate, revealing novel biochemical pathways of toxicity. The study highlights the possibility of using metabonomics in hESCs and hESC-derived cells as a novel tool to identify predictive biomarkers for efficacy and safety assessment of pharmaceuticals (47). In 2010 the same group provided a more comprehensive dataset in which 7 out of 8 substances were correctly classified (48).

However, many challenges remain before the full potential of metabonomics in in vitro toxicology will be developed. One major challenge is the identification of masses: approximately fifty percent of the measured masses are not annotated in public databases. Deciphering chemical structures to identify small molecules is labor-intensive and time-consuming, requiring costly NMR spectroscopy and/or MS-MS setups that are not generally available to most biomedical research laboratories.

4.3. Toxicoproteomics

Another approach to identify biomarkers for developmental toxicity is based on proteomics technology. Toxicoproteomics integrates the traditional toxicology and pathology with differential protein and gene expression analysis and systems biology. It focuses mainly on the assessment of the proteome in organs and biofluids, such as liver and blood. Toxicoproteomics is mainly used to discover key modified proteins as early biomarkers for the prediction of an adverse effect. In addition, the knowledge of the perturbed pathway will provide the necessary mechanistic support when interpreting the data of animal studies and their relevance for humans (67). The identification of novel biomarkers and their mechanistic interpretation is still time consuming, however, using high density LC-MS/MS platforms and its complementary methods can accelerate the toxicoproteomics analysis.

Beside the wide use of toxicoproteomics for diagnosis in clinical chemistry, or in the identification of biomarkers for specific environmental exposures or stressors, it can also play a role in in vitro toxicology studies. Groebe et al. have challenged the validated murine embryonic stem cell test with two known embryotoxicants (warfarin and lovastatin) in order to further evaluate the mode of action which seems to be related to the Ras signalling pathway (68). However, further studies are necessary to confirm the relevance of identified proteins as biomarkers. Because toxicoproteomics have the potential to establish a relationship between adverse effects and protein markers they have the great potential to contribute to an understanding of toxicological mechanisms.

5. PERSPECTIVE

Some human endpoints in toxicity testing are suffering from low concordance between animal studies and humans (e.g. cardiotoxicity, hepatoxicity, developmental toxicity, metabolism-mediated toxicity). For this reason toxicity tests that are based on human stem cells have been developed in the recent years to overcome the interspecies variations. Another challenge in toxicity testing is the need for medium to high throughput which can be facilitated by the unlimited supply by stem cells. Human stem cells have some other advantages because they are of non-malignant origin and can generate several types of cells of toxicological relevance.

However, the major bottlenecks in the development of such in vitro toxicity tests remain the differentiation protocols, leading to an insufficient number of toxicologically relevant target cells. In the past, several EU-funded projects have addressed the development of stem cell-based in vitro tests e.g. ReProTect, InVitroHeart and Vitrocellomics. ReProTect allowed the identification of new valuable in vitro assays that can partially mimic the complexity of the mammalian reproductive cycle. Several of the developed tests are now considered for the entry into prevalidation. The aim of the InVitroheart project was to establishing relevant hES cell derived cardiomyocytes cultures whereas the Vitrocellomics project focused on stem cell derived hepatocytes to be used for drug discovery and toxicity testing. Additional support might come from other international R & D collaborations in the field of regenerative medicine aiming to develop cell-based therapies, since some target cells for toxicological testing are also of therapeutic interest. In addition, basic research activities may contribute to the optimization of differentiation conditions (69). It is also expected that emerging technologies in cell culture such as 3D models, supporting biomaterials, etc. will contribute to the upscaling of toxicological relevant cells.

Nevertheless, scientific consensus on the degree of functionality of the in vitro system and/or on the expression levels of a panel of marker genes of the relevant target cells is urgently required in order to define the maturation stage of the cell types of interest as well as the level of purity/homogeneity of cultures used for toxicity assessments. This information will underline the correlated biological relevance of cellular models based on stem cells or their derivates. Induced pluripotent stem cell technology might be able to provide an unlimited cell supply and may have other advantages such as the possibility to generate cells for different genetic backgrounds and disease models. At a later stage in the development of a test, the use of iPSCs and their derivates may gain international acceptance in relevant test guidelines for hazard identification. However, the technologies based on iPSCs are still in their infancy and need to be further developed. Nevertheless, these activities require a link to well-characterized standard models which can currently only be provided by hESCs.

The selection of predictive readout systems depends mainly on the purpose of the tests. While in the area of cardiotoxicity and neurotoxicity multielectrode arrays are well established, and promising (with feasibility studies ongoing); other areas such as the area of developmental toxicity still suffer from the lack of suitable predictive biomarkers, which is clearly related to the complexity of the field (Figure 2). Emerging technologies, such as the ones discussed in this review, will hopefully provide input to this scientific challenge. In addition, several strategies are currently being defined combining various stem cell-based in vitro tests with genomic readouts. These strategies aim at improving the developmental toxicity predictions by identifying new relevant biomarkers. This strategy will be further complemented by metabolic mediated toxicity systems and PBPK modeling in order to provide a better in vitro toxicity-based hazard identification and characterization for this particular application.

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http://dx.doi.org/10.4049/jimmunol.0803670
PMid:19454684

Abbreviations: a1-AT- alfa 1 antitrypsin, AAT- alfa 1 antitrypsin, AcH- acetylcholine, AcHE- acetylcholine esterase, ACTC- alpha cardiac actin, ACTN2- alpha actinin 2, AFP- alfafetoprotein, ALB- albumin, APOF- apolipoprotein F, ARG- arginase, ASGPR- asialoglycoprotein receptor, BAX- Bcl- associated X protein, BCL2- B cell lymphoma 2, BDNF- brain- derived neurotrophic factor, BF- brain factor, BMP- bone morphogenetic protein, BRN- POUF, C/EBP- CCAAT/enhancer binding protein, C3, C5- complement component 3, 5, cAMP- cyclic AMP, CASP3- caspase 3, CC16- secretoglobim, CCDN1- cyclin D1, CCRK- cyclin dependent kinase, CEBPA- CCAAT/enhancer binding protein, CFABP- fatty acid binding protein, CK- keratin, C-KIT- cellular homolog of the feline sarcoma viral oncogene v-kit, CNTF-ciliary neurotrophic factor, CXCR- chemokine (C-X-C motif) receptor, CYP- cytochrome P450, DAZ- deleted in azoospermia, DAZL- deleted in azoospermia-like, DMEM- Dulbecco's modified Eagle's media, DMSO- dimethylsulfoxide, DNMT3B- DNA (cytosine-5-)-methyltransferase 3 beta, DPPA (STELLA)- developmental pluripotency associated, EB- embryoid body, EGF- epidermal growth factor, EPA- Environmental Protection Agency, EROD- ethoxyresorufin-O-deethylase, EtOH- ethanol, FBS- fetal bovine serum, FOX- forkhead box, FSHR- follicle stimulating hormone receptor, GAPDH- glyceraldehyde-3-phosphate dehydrogenase, GATA4- GATA binding protein 4, GCTM2- cell surface keratan sulfate, GDNF- glial cell derived neurotrophic factor, GFAP- glial fibrillary acidic protein, GFP- green fluorescent protein, GST- glutathion- S- transferase, HB9- motor neuron and pancreas homeobox 1, HCN- hyperpolarization activated cyclic nucleotide-gated potassium channel, HERG- potassium voltage-gated channel, HEX- hematopoietically expressed homeobox, HGF- hepatocyte growth factor, HNF4- hepatocyte nuclear factor 4, HOX- homeobox, HPPA- atrial natriuretic factor, ICG- indocyanin green, IFITM3- interferon induced transmembrane protein 3, Ikr- potassium voltage-gated channel, ISLET- islet amyloid polypeptid, K18- keratin 18, KCNH2- potassium voltage-gated channel H2, KRT- keratin, lama3- laminin alpha 3, LDH- lactate dehydrogenase, LDL- low density lipoprotein, LHX- LIM homeobox, MAP- mitogen activated protein, MASH1- achaete-scute complex homolog 1, MEA- multielectrode array, MIRP- potassium voltage-gated channel, Isk-related family, MIS- Mullerian Inhibiting Substance, MLC- megalencephalic leukoencephalopathy with subcortical cysts, MLH1- mutL homolog 1, MPP+- 1-methyl-4-phenylpyridinium, MRP- multidrug resistance-associated protein, MSX1- msh homeobox 1, MYH6- myosin heavy chain 6, MYL- myosin light chain, NANOG- nanog homeobox, N-cad- neuronal cadherin, NCAM- neural cell adhesion molecule, NEFH- neurofilament heavy polypeptide, NEFL- neurofilament light polypeptide, NES- nestin, NEUROD- neurogenic differentiation, NINDS- National Institute of Neurological Disorders and Stroke, NKX- NK homeobox, NP- neural precursors, OATP2- solute carrier organic anion transporter family, OCT4- POU class 5 homeobox, OLIG- oligodendrocyte transcription factor, OSM- oncostatin M, PAX- paired box, penG- penicillin G, PEPCK- phosphoenolpyruvate carboxykinase, PI3- phosphoinositide-3-kinase, PIWIL2- piwi-like 2, PLN- phospholamban, POU5F1- POU class 5 homeobox 1, PRDM- PR domain containing, PUM2- pumilio homolog 2, RA- retinoic acid, ROS- reactive oxygene species, RT-PCR- real time PCR, SCP- sterol carrier protein, SHH- sonic hedgehog, SMI- hypophosphorylated neurofilament, SMN- survival of motor neuron, SOX- sex determining region Y-box, SPC- surfactant protein C, SSEA- stage specific embryonic antigen, SYCP- synaptonemal complex protein, T- T bracyury homolog, TAT- tyrosine aminotransferase, TBPA- thyroxine-binding prealbumin, TCR- T cell antigen receptor, TDGF- teratocarcinoma-derived growth facto, TEKT- tektin, TF- tranferrin, TH- tyrosine hydroxylase, TNNT2- troponin T type 2, TO- tryptophan dioxygenase, TPH- tryptophan hydroxylase, TTX- tetrodotoxin, TUBB3- tubulin beta 3, TUJ1- Neuron-specific class III beta-tubulin, UDP- uridine diphospho, VASA- DEAD box protein, VE-cad- vascular endothelial cadherin, WNT- wingless-type MMTV integration site family, WT1- Wilms tumor 1, ZIC1- zinc finger protein, ZIPRO- zinc finger and SCAN domain-containing protein

Key Words: Human pluripotent stem cells, In vitro toxicity testing, Test development criteria, Review

Send correspondence to: Susanne Bremer-Hoffmann, Systems toxicology unit, ECVAM, European Centre for the Validation of Alternative Methods, T.P. 580, IHCP, JRC, European Commission, via E. Fermi 2749, 21027 Ispra,VA, Italy, Tel: 39 0332 785914,
Fax: 39 0332 785336, E-mail: susanne.bremer-hoffmann@ec.europa.eu