Human embryonic stem cells (hESC) are now routinely cultured in many laboratories, and differentiation protocols are available to generate a large variety of cell types. In an ongoing ethical debate opinions of different groups are based on varying sets of religious, historical, cultural and scientific arguments as well as on widely differing levels of general information. We here give an overview of the biological background for non-specialists, and address all issues of the current stem cell debate that are of concern in different cultures and states. Thirty-five chapters address embryo definition, potential killing and the beginning of human life, in addition to matters of human dignity, patenting, commercialisation, and potential alternatives for the future, such as induced pluripotent (reprogrammed) stem cells. All arguments are compiled in a synopsis, and compromise solutions, e.g. for the definition of the beginning of personhood and for assigning dignity to embryos, are suggested. Until recently, the major application of hESC was thought to be transplantation of cells derived from hESC for therapeutic use. We discuss here that the most likely immediate uses will rather be in vitro test systems and disease models. Major and minor pharmaceutical companies have entered this field, and the European Union is sponsoring academic research into hESC-based innovative test systems. This development is supported by new testing strategies in Europe and the USA focussing on human cell-based in vitro systems for safety evaluations, and shifting the focus of toxicology away from classical animal experiments towards a more mechanistic understanding.

The potential health effects of inhaled engineered nanoparticles are almost unknown. To avoid and replace toxicity studies with animals, a triple cell co-culture system composed of epithelial cells, macrophages and dendritic cells was established, which simulates the most important barrier functions of the epithelial airway.
Using this model, the toxic potential of titanium dioxide was assessed by measuring the production of reactive oxygen species and the release of tumour necrosis factor alpha. The intracellular localisation of titanium dioxide nanoparticles was analyzed by energy filtering transmission electron microscopy. Titanium dioxide nanoparticles were detected as single particles without membranes and in membrane-bound agglomerates. Cells incubated with titanium dioxide particles showed an elevated production of reactive oxygen species but no increase of the release of tumour necrosis factor alpha.
Our in vitro model of the epithelial airway barrier offers a valuable tool to study the interaction of particles with lung cells at a nanostructural level and to investigate the toxic potential of nanoparticles.

In hepadnaviruses, reverse transcription is primed by the viral reverse transcriptase (RT) and requires the specific interaction between the RT and the viral RNA encapsidation signal termed ε. To study the activity of the RT in vitro, the current procedure uses in vitro translated duck hepatitis B virus polymerase, but not the hepatitis B virus polymerase itself, in the rabbit reticulocyte lysate expression system.
Here, the hepatitis B virus (HBV) polymerase has been successfully expressed in a translational extract that was obtained from monolayer human hepatocyte cells HuH-7. The translated polypeptide retained the RNA-directed polymerase (reverse transcriptase) activity on the viral RNA template containing the ε signal. We suggest that the reverse transcription event of the viral RNA coding for the polymerase and containing an ε structure is concomitant to the translation of the viral polymerase to the messenger RNA. In contrast to the duck polymerase, only a fraction of the reverse transcribed complementary DNA (cDNA) was covalently bound to the HBV polymerase in this system. When the ε signal was missing on the mRNA, the translated full-length HBV polymerase could not reverse transcribe the viral RNA template. A truncated HBV polymerase that was lacking the YMDD catalytic active site for the initiation of reverse transcription was unable to reverse transcribe the viral mRNA template containing the ε signal. The reverse transcription activity could also be partially inhibited by employing nucleoside analogues, such as 2’-3’-dideoxy-3’-thiacytidine (3TC; lamivudine) in the expression system.
The procedure described here provides a method for the in vitro screening of new anti-HBV compounds directed against wildtype and mutants of this crucial viral protein, the HBV polymerase, without the use of animals (ducks) or animal extracts (rabbit reticulocyte lysate).

Laboratory animal models are limited by scientific constraints on human applicability, and increasing regulatory restrictions, driven by social concerns. Reliance on laboratory animals also incurs marked – and in some cases, prohibitive – logistical challenges, within high-throughput chemical testing programmes, such as those currently underway within Europe and the US. However, a range of non-animal methodologies is available within biomedical research and toxicity testing. These include: mechanisms to enhance the sharing and assessment of existing data prior to conducting further studies, and physicochemical evaluation and computerised modelling, including the use of structure-activity relationships and expert systems. Minimally-sentient animals from lower phylogenetic orders or early developmental vertebral stages may be used, as well as microorganisms and higher plants. A variety of tissue cultures, including immortalised cell lines, embryonic and adult stem cells, and organotypic cultures, are also available. In vitro assays utilising bacterial, yeast, protozoal, mammalian or human cell cultures exist for a wide range of toxic and other endpoints. These may be static or perfused, and may be used individually, or combined within test batteries. Human hepatocyte cultures and metabolic activation systems offer potential assessment of metabolite activity and organ-organ interaction. Microarray technology may allow genetic expression profiling, increasing the speed of toxin detection, well prior to more invasive endpoints. Enhanced human clinical trials utilising microdosing, staggered dosing, and more representative study populations and durations, as well as surrogate human tissues, advanced imaging modalities and human epidemiological, sociological and psychological studies, may increase our understanding of illness aetiology and pathogenesis, and facilitate the development of safe and effective pharmacologic interventions. Particularly when human tissues are used, non-animal models may generate faster, cheaper results, more reliably predictive for humans, whilst yielding greater insights into human biochemical processes. Greater commitment to their development and implementation is necessary, however, to efficiently meet the needs of high-throughput chemical testing programmes, important emerging testing needs, and the ongoing development of human clinical interventions.

A survey of publicly funded research specifically targeting alternatives to animal testing was conducted over 2006/2007. Responses were received from 16 European countries (Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Hungary, Italy, Netherlands, Norway, Slovakia, Spain, Sweden, Switzerland and the United Kingdom). The responses were compiled by national agencies or national consensus platforms. The current annual total across the 16 countries was estimated as € 17 million. The largest contribution came from Germany with € 4.6 million (27% of the total). Also collated was information on the existence of a national strategy on alternatives research, the focus of any such strategies, the research priority setting process, stakeholder consultation in that process, project funding preferences or limits, coordination mechanisms and the separation of responsibilities of competent authorities (i.e. for research support, laboratory animal welfare and chemicals management). Countries with national strategies (France, Germany, the Netherlands, Sweden, Switzerland and the UK) are skewed towards the higher end of the spending distribution. These 6 countries account for over € 12 million, i.e. >70% of the overall total of national spending identified. Most countries have national consensus platforms. These should help to both stimulate stakeholder consultation and further national spending on alternatives research. The situation regarding the separation of responsibilities of competent authorities (i.e. for research support, laboratory animal welfare and chemicals management) is mixed. A degree of overlap exists in many cases. A research strategy that is receptive to and reflects regulatory developments – such as REACh with its marked resultant increase in animal use – is an obvious need that is as yet unmet in many of the countries surveyed. The need for a mechanism to collate details of active research projects within Europe as a whole was also identified.