Efficacy and Mode of Action Studies Systems Biology Using Embryonic Stem Cell Based Screening Systems

In terms of efficacy, a variety of in-vitro studies employing mESC for testing neuroprotective compounds and conditions have been published. Essentially, the protocols have been adapted from corresponding experiments using primary cultures [72]. Specific agonists and antagonists allow fairly precise pharmacological characterization ofthe major ionotropic and metabotropic receptors (for glutamate,

GABA, acetylcholine, etc.) and voltage-dependent ion channels in neural cell culture models [72]. The precise control of these physiological responses is prerequisite for defining appropriate experimental windows for the subsequent molecular analysis or pharmacological profiling. For a replacement of animal models for neuroprotection, essentially a variety of cellular insults are performed (such as excitotoxic, ischemic or by oxidative or P-amyloid-related stress). Neuroprotective conditions are then monitored on the levels of molecular markers from differential protein analysis and functional read-outs. The simplest read-out is quantification of neuronal survival by functional vitality controls, fluorescent vital staining, or live cell staining. As shown in Figure 8.5, it follows correlational analysis of functional and molecular data, and the definition of physiological and/or pharmacological/toxic endpoints.

The relevant cell status for effective neurotoxic conditions is defined by the outcome of dose-response curves in calcium-signaling experiments; rescue by neuroprotective conditions quantified in terms of surviving neurons, etc. ESC are thus embedded in a seamless and iterative process, where emerging molecular surrogates can be fed back directly to functional in-vitro systems (Validation I), and fed forward to in-vivo models generated by using the very same ESC (Validation II), under comparable paradigms ofintervention (for example, siRNA). Moreover, conditions tested in neural derivatives can be checked in genetically homogeneous alternative differentiation protocols (Validation III; for example, does a neuroprotective substance have adverse effects in hepatocytes?). ESC models offer the huge advantage of avoiding genetic variation, one of the major factors complicating biomarker validation.

By summarizing the examples in Figure 8.5, the authors would like to emphasize two important points:

Next to a characterization of tissue-specific differentiated endpoints from ESC by molecular markers, a thorough functional characterization and control of corresponding cell cultures is absolutely mandatory. In the case ofneurons, efficacy studies need to be performed in a context of pharmacological and physiological consistency.

In contrast to strategies employed in the therapeutic field, "pure" endpoints (e.g., solitary neurons for transplantation) are often far too artificial and short-lived and, moreover, ignore epigenetic events and reprogramming [73].

Thus, the use of hESC in-vitro models on the contrary aims at generating stable cell culture conditions, including mixed cultures with multiple cell types which, in some cases, provide a better match of organotypic "physiological" conditions. Mixed neural cell cultures with about 15% neurons and a variety of supporting cells, for example, much more closely represent the situation in a brain. In the case of neurons, the basic requirement would be the presence of major neuronal ion channels (e.g., NMDA-, GABAa- and cholinergic receptors) in appropriate proportions and with adequate pharmacology. These neural cultures could only then be used as models for important human diseases of the nervous system. The corresponding molecular analysis of functional mESC models for excitotoxicity and ischemia, consequently revealed surrogate biomarkers such as superoxide

Fig. 8.5 Embryonic stem cell (ESC)-based in-vitro systems for neurotoxicity and neuroprotection screening. In a context of cutting-edge analytical technologies, these offer the opportunity to integrate functional read-outs (toxicology-, mode of action- or efficacy-related) with precisely controlled biomarker signatures (proteins, metabolites, or other effectors). The corresponding data mining (mostly automated) generates contextual information from chemical, genetic or literature databases (Bioinformatics II), which in an essentially iterative process can instantly be fed back into experimental procedures or data layers. On a first level (Bioinformatics I), mass spectrometry-based investigation of chemical details underlying differential molecular information produces a refined mechanistic understanding of the functional read-outs. Emerging key surrogate markers can be directly fed back into in-vitro and in-vivo models (Validations I and II) by, for example, hypothesis-driven silencing technologies (siRNA). ESC-based in-vitro models (either murine or human) have one huge advantage, namely that they can be used directly to generate corresponding in-vivo models. A further huge advantage is the possibility to test, for example, neuroprotective substances in genetically homogeneous different organ-specific cell types, such as hepatocytes or cardiomyocytes (Validation III). The mechanistic information form (e.g., differential proteomic profiling) helps to refine the control and read-outs from related in-vivo models (Western blots, chip-based markers, pharmacology). The combined functional and molecular information of this type can help to refine chemical concepts of intervention (quantitative structure-activity relationship (QSAR), molecular modeling).

dismutase (SOD), neuregulins or dihydropyrimidinase-related protein 2 (DRP-2) [28, 72, 74], which are consistently implicated independently by genomic analysis in human neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease, and others [75].

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