Taking the Stem Cell Debate to the Public

In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholder

Markers of Murine Embryonic and Neural Stem Cells, Neurons and Astrocytes: Reference Points for Developmental Neurotoxicity Testing

Developmental neurotoxicity (DNT) is a serious concern for environmental chemicals, as well as for food and drug constituents. Animal-based DNT models have relatively low sensitivity, and they are limited by high work-load, cost and animal ethics. Murine embryonic stem cells (mESC) recapitulate several critical processes involved in the development of the nervous system if they are induced to differentiate into neural cells. They therefore represent an alternative toxicological model to predict human hazard. In this review, we discuss how mESC can be used for DNT assays. We have compiled a list of mRNA markers that define undifferentiated mESC (n = 42); neural stem cells (n = 73), astrocytes (n = 25) and the pattern of different neuronal and non-neuronal cell types generated (n = 57). We propose that transcriptional profiling can be used as a sensitive endpoint in toxicity assays to distinguish neural differentiation states during normal and disturbed development. Importantly, we believe that it can be scaled up to relatively high throughput whilst still providing rich information on disturbances affecting small cell subpopulations. Moreover, this approach can provide insight into underlying mechanisms and pathways of toxicity. We broadly discuss the methodological basis of marker lists and DNT assay design. The discussion is put in the context of a new generation of alternative assays (embryonic stem cell based DNT testing = ESDNT V2.0), that may later include human induced pluripotent stem cells, and that are not designed for 1:1 replacement of animal experiments, but are rather intended to improve human risk assessment by using independent scientific principles.JRC.I.2-Validation of Alternative Method

Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the 'human embryonic stem cell (hESC)- derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (\20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large 'common response' to VPA and MeHg could be distinguished from 'compound-specific' responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.EU/FP7/ESNATSDFGDoerenkamp-Zbinden Foundatio

Vor- und Nachdenkliches ... zum wahren Erfolg des 3R-Prinzips

Ist es möglich, den "Wert" einer wissenschaftlichen Disziplin zu bemessen? Wie kann diese Disziplin dann in einem Gesamtrahmen eingeordnet werden, und nicht zuletzt, ist eine solche Überlegung überhaupt gerechtfertigt? Von einigen Seiten mögen bei solchen Fragen Zweifel aufkommen; andere werden die Forschung im Bereich der Alternativmethoden zu Tierversuchen als kategorischen Imperativ ohne jegliches wenn und aber betrachten. Schließlich gibt es noch eine etwas differenziertere Sichtweise auf diese Problematik, die sehr häufig von Personen in gesetzgebender Funktion, von privaten wie öffentlichen Geldgebern, Tierschutzbeauftragten, und nicht zuletzt von jungen Studierenden, die sich entschließen, ihre Karriere im Bereich der Alternativmethoden zu beginnen, vertreten wird. Diese unterschiedlichen Betrachter stellen früher oder später eine Kosten-Nutzen Rechnung auf und wägen verschiedene Aspekte gegeneinander ab. Mit diesem Ansatz stehen sie jedoch nicht alleine da: schließlich wird ein Großteil der Steuerzahler und Wähler ähnliche Gedanken anstellen. Aus diesem Grund erscheint es mehr als gerechtfertigt, einige grundsätzliche Gedanken über den bereits erwähnten Wert des 3R Ansatzes (Replace, Reduce, Refine; Ersetzen, Reduzieren, Verbessern), wie er von Russell und Burch 1959, sowie in der Bologna-Erklärung 1999 aufgestellt wurde, zu formulieren. Nach rein qualitativen Gesichtspunkten wird die wachsende Bedeutung von Alternativmethoden schon durch die Einrichtung einer eigenen Institution (ECVAM), deren Aufgabe die Etablierung und Bewertung alternativer in vitro Methoden ist, durch die EU unterstrichen. Ferner stützt sich bereits ein Großteil der neueren EU-Gesetzgebung (REACH) auf die Verwendung von Alternativmethoden (REACH, 2006). In diesem Zusammenhang arbeitet die EU-Kommission bereits seit einiger Zeit in enger Partnerschaft mit der Industrie (EPAA, 2007), und zusätzlich wurden an Universitäten neue Lehrstühle für die Entwicklung von Alternativmethoden zu Tierversuchen eingerichtet (Leist, 2006; Wendel, 2002). Eine Reihe quantitativer Versuche, die Entwicklung von Alternativmethoden zu erfassen, stützt sich als alleinige Bewertungsgrundlage auf den quantitativen Tierverbrauch innerhalb der EU oder in den einzelnen Mitgliedsstaaten. Ferner wurde auch die Anzahl der neuen OECD-Richtlinien, die Alternativmethoden als Evaluationsmethode beinhalten, statistisch erfasst und zur Auswertung herangezogen. Basierend auf diesen Zahlen erscheint der Fortschritt in der Implementierung des 3R-Prinzips relativ langsam und wenig bedeutsam. Es ist uns daher ein besonderes Anliegen an dieser Stelle klar hervorzuheben, dass durch eine solche Herangehensweise die wahren Erfolge und Fortschritte, die bereits im Bereich der Entwicklung von Alternativmethoden erzielt worden sind, bei weitem unterbewertet wurden. Aus diesem Grund möchten wir im Folgenden einige Denkfehler diskutieren, die regelmäßig in der öffentlichen Debatte auftreten und die in großem Maße zu der bereits erwähnten Unterbewertung des 3R-Ansatzes geführt haben

Food for Thought ... on the Real Success of 3R Approches

Can the value of a scientific discipline be gauged? Where does the discipline stand? Is such a consideration important at all? Some may have doubts about the usefulness of asking such questions. Some will see the research into alternative methods as a categorical imperative. Others again will have a more differentiated view, a group that will comprise policy makers, those that provide funding and infrastructure for research, those that are responsible for our safety and that of our environment, and, last but not least, those that are about to choose their future field of scientific work. All these person groups will at some point use cost-benefit considerations and value balances. This type of thinking also applies largely to the general population of tax payers and voters. For this reason, some thoughts on the real value of 3R approaches (replace, reduce, refine, as defined by Russell and Burch, 1959, and the declaration of Bologna, 1999 (3R, 2004)) appear to be justified and necessary. On a qualitative level, the value of alternative methods is underscored by the fact that the EU funds an entire research institution (ECVAM) dedicated solely to the evaluation of alternative methods, and that major new EU legislation, such as REACH, has a strong focus on the use of alternative methods (REACH, 2006). Moreover, industry and the European Commission work together in a partnership for alternative approaches (EPAA, 2007), and universities are starting to establish departments dedicated to 3R research (Leist, 2006; Wendel, 2002). More quantitative approaches to describe the success of the new field make use of the statistics of animal use in the EU or its individual member states, or they count the number of OECD test guidelines that rely on alternative assays for safety evaluations. On this basis, progress of 3R is sometimes described as being relatively slow. In our opinion, such strategies to gauge the success of alternative approaches largely underestimate the real success of this emerging research field. Therefore, we will highlight in the following a number of conceptual errors that contribute to the underestimation of the value of 3R, and that are frequently encountered in public discussions

Protein kinase PKR is required for platelet-derived growth factor signaling of c-fos gene expression via Erks and Stat3

The double-stranded RNA (dsRNA)-activated protein kinase PKR is an interferon (IFN)-induced enzyme that controls protein synthesis through phosphorylation of eukaryotic initiation factor 2α (eIF-2α). PKR also regulates signals initiated by diverse stimuli, including dsRNA, IFN-γ, tumor necrosis factor-α, interleukin-1 and lipopolysaccharide, to different transcription factors, resulting in pro-inflammatory gene expression. Stat3 plays an essential role in promoting cell survival and proliferation by different growth factors, including platelet-derived growth factor (PDGF). Here we show that PKR physically interacts with Stat3 and is required for PDGF-induced phosphorylation of Stat3 at Tyr705 and Ser727, resulting in DNA binding and transcriptional activation. PKR-mediated phosphorylation of Stat3 on Ser727 is indirect and channeled through Erks. Although PKR is pre-associated with the PDGF β-receptor, treatment with PDGF only modestly activates PKR. However, the induction of c-fos by PDGF is defective in PKR-null cells. Taken together, these results establish PKR as an upstream regulator of activation of Stat3 and as a common mediator of both growth-promoting and growth-inhibitory signals