Reproductive Toxicology and hESC

There is a worldwide dedicated research effort towards the continuous improvement of risk assessment and health and safety protection. In the European Commission's regulatory framework of drug approval for treatments of patients, certain sets of standardized animal tests are performed according to the European Council directives [43-45] and the guidelines of the International Conference on Harmonisation [46]. The hierarchical decision-tree approaches defined by the European Commission [47] and the Organisation for Economic Cooperation and Development [48, 49] for international standards of pharmacological and toxicological tests primarily address the adult organism. Currently, although comprehensive multigenerational studies are undertaken to provide information about all aspects of reproductive and developmental toxicity, developmental studies to address the specific risks of the developing embryo are not regulatory requirements and, therefore, appropriate methods for the screening of toxic substance effects during embryonic development (embryo- and fetal toxicity) are essentially absent. For example, there were cases of important toxic side effects with pathological consequences during development, which were only discovered after approval for therapeutic applications in patients (the dire case of

Contergan). Likewise, more than 30 compounds with highly toxic potential had to be withdrawn from the market by the FDA (Food and Drug Administration) during the years 1998 to 2001, in each case without being detected by the required set of prior animal tests (one of the most recent examples was Lipobay). Among these 30 drugs, eight caused substantial side effects, especially in women. Current animal tests are simply not sufficient to detect gender-specific predispositions in humans. Moreover, during the time after Contergan, about 85% of those cases of developed drugs with highly toxic potential could not be predicted safely by animal tests [50, 51].

For human embryotoxicity, and especially in terms of neurodevelopmental processes, the question is whether the use of human embryonic or adult stem cells is better to establish in-vitro screening methods. Basically, stem cells have two characteristic properties: (1) they can be maintained undifferentiated in culture for long periods of time; and (2) they can be induced into specific organotypic differentiations. As shown in Figure 8.4, there exist both embryonic and adult tissue-specific stem cells. Embryonic stem cells are established from the inner cell mass (ICM) of 5- to 7-day-old blastocysts [52, 53]; the tissues of origin for adult stem cells are marked in red.

For the long-term cultivation of undifferentiated cells, ESC cultures are maintained in co-culture with so-called "feeder" cells from mouse fibroblasts [54], or cell-free in conditioned media [55]. The major inhibitory factor for differentiation in the murine cell culture system is the cytokine leukemia inhibitory factor (LIF) [56], while the adequate factor for long-term hESC culture is still under investigation. ESC display far-reaching pluripotent properties; for example, they can differentiate into virtually every organ-specific human cell type when cultured as embryoid bodies. Differentiation into diverse cell types of endodermal (e.g., pancreatic and hepatic cells), mesodermal (e.g., bones, muscle and blood cells, cardiomyocytes) or ectodermal origin (e.g., neurons and glial cells) is regulated by LIF deprivation for mESC, and the proper combination of growth factors. The published reports on the differentiation potential of mESC and hESC are summarized in Table 8.1.

Adult stem cells are derived from mature organs of adult individuals such as the epidermis, hair, intestine, liver, hematopoietic system, brain or bone marrow, and have limited possibilities of proliferation and differentiation ("developmental restriction"). Due to their multipotent properties, these stem cells are mostly restricted to one tissue only. The plasticity of the adult stem cells - which means the ability to contribute into cell types characteristic of another organ - was only demonstrated for hematopoietic stem cells, stromal cells of the bone marrow and multipotent adult precursors, which can be generated in vitro from certain cells of the bone marrow and certain neural cells [57] (see also Fig. 8.3). The question of whether adult stem cells can be artificially redifferentiated is currently under intense investigation, but remains open for the time being. A further disadvantage of adult stem cells is their limited ability to differentiate and proliferate, which goes together with decreased lifetime in vitro. Moreover, only very small numbers of these cells are available (they decrease even further with age), they are difficult

Fig. 8.4 An overview of adult and embryonic stem cells. Starting from the blastocyst stage, the inner cell mass (ICM) is used for the generation of embryonic stem cell (ESC) lines. These ESC lines can be maintained and expanded easily, are pluripotent, and can be differentiated to all types of tissue-specific cell lines of the three germ layers under

Fig. 8.4 An overview of adult and embryonic stem cells. Starting from the blastocyst stage, the inner cell mass (ICM) is used for the generation of embryonic stem cell (ESC) lines. These ESC lines can be maintained and expanded easily, are pluripotent, and can be differentiated to all types of tissue-specific cell lines of the three germ layers under appropriate conditions (see Table 8.1). In contrast, adult/somatic stem cells derived from tissues of adult organisms are more likely multipotent and, depending on their origin, they can only differentiate to a limited set of endpoints. Moreover, they are difficult to obtain and cannot be maintained and expanded in large-scale quantities.

to distinguish from surrounding cells, and their derivation is either difficult or impossible, depending upon the original organ. To date, it remains unclear as to how adult stem cells function and how their potential might be optimally exploited [58-60].

Consequently, results with direct relevance for human developmental toxicity can only be obtained using hESC approaches. Only these models can provide information about the effects of compounds on all aspects of embryonic neuronal development.

Now, using hESC, there exists for the first time the chance to develop innovative in-vitro analytical methods for the early detection of human-specific embryotoxic risks of compounds and chemicals. The combination of hESC-based in-vitro test models with cutting-edge molecular analysis holds the promise to deliver - with extreme sensitivity - highly relevant functional and molecular marker endpoints for the early detection of embryotoxic effects of drugs or chemicals. The outstanding potential of hESC-based in-vitro models has thus to be viewed in context with results from corresponding mESC-screening systems. In combination with quantitative differential proteomic display techniques, biomarkers for neurotoxicity have already been explored. A variety of ESC-based tests have already been validated and are in use for regulatory purposes [39-42, 61]. In terms of relevance, proteomics is in principle far superior to RNA/DNA-based array technologies, because the relatively static and small human genome of approximately 20 000 genes is translated into a highly dynamic and complex proteome of several million protein molecules of post-translationally modified isoforms. It is on this latter level where dynamic functional aspects of cellular events take place [22, 62-68]. Both neurotoxic and embryotoxic substances bind to proteins, changing interactions in complex functional networks, sometimes on very rapid time scales (e.g., phosphorylation), and the changes induced by this type of intervention are ideally analyzed by proteomics technologies. The hope is that emerging molecular signatures from mESC can in the future also be derived from functionally controlled differentiated hESC, with key biomarker proteins identified and quantified, thus enabling a molecular understanding of side effects and innovative strategies for acceleration in drug development, validation, and toxicity testing [69].

A clear understanding of the neurotoxic effects of medical drugs for human embryos during pregnancy would constitute a major contribution towards improved drug safety and preventive healthcare. The effects of neurotoxic substance application can be defined on the level ofdistinct molecular consequences in terms of immediate protein expression changes. The advanced proteomics technologies mentioned above, in correlation with synchronized functional/physiological measurements, can provide a comprehensive, precise and quantitative molecular pattern analysis of the underlying mechanisms. Moreover, the availability of human biomarkers for neuronal embryotoxicity could drastically improve early screening procedures for embryotoxic and neurotoxic substances.

A number of projects are ongoing, with hESC being cultivated in vitro and differentiated to functional neurons according to published procedures. Undifferentiated hESC are characterized by surface antigens such as SSEA-3, SSEA-4 [70], TRA-1-60, TRA-1-81, Oct-4 [71] and GCTM-2; or by enzyme activities (alkaline phosphatase). Neurons differentiated from precursors are characterized by morphological criteria and immunohistochemistry (antibodies for neuron-specific proteins MAP-2 and synaptophysin). Subsequently - and more importantly - a functional and physiological control of neurons by their response to various neuron-specific stimuli (neurotransmitter, depolarization, etc.) is performed and measured by, for example, single cell Ca-imaging.

At present, the main objective ofrelated projects is the identification ofmolecular mechanisms underlying the human neurotoxicity of 20 selected substances [5, 37]. The corresponding events take place in early embryonic and specifically neural developmental stages and maturation processes. Thus, an in-vitro test system based on hESC is ideally suited for corresponding functional and molecular analysis. Cells will be exposed to various concentrations of toxic compounds and analyzed on the molecular level at various endpoints, representing precursors, early and mature stages of neurons [5]. Dose-response relationships of toxic effects induced by substances are essentially quantified by the influence of these compounds on normal functional signals measured by calcium imaging. Even subtle influences of toxic compounds upon intracellular transient calcium concentration changes due to normal reactions caused by neurotransmitters or other physiological parameters will become apparent on a statistically significant level.

Based on the functional analysis, protein pellets will be generated from treated and untreated neuronal differentiated human stem cells at appropriate endpoint conditions, and subsequently be submitted to a quantitative and differential protein pattern analysis (see Fig. 8.2).

Under the given circumstances ofethical controversy concerning hESC and strict legal guidelines for stem cell importation and use in the European Community (in particular in Germany), it is very sensible to set standards with certain registered and controlled hESC lines (http://stem.nih.gov/research/registry). Arguably, this would not lead to a proliferate generation of ever new and individualized cell lines with associated scarification of embryos, as in the case of therapeutic stem cell approaches, but on the contrary, it would lead to the development of one or a few existing lines for very broad and standardized application [21]. In this way, it is conceivable for the first time to exploit the outstanding features of hESC and establish related innovative screening methods for embryotoxicity and neurotoxicity, including the identification of molecular toxicity biomarkers without using animal-based in-vitro or in-vivo systems. Moreover, it would avoid some of the burning ethical issues haunting the therapeutic branch of hESC research.

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