Towards the development of an in vitro assay to predict osteotoxicity in the developing embryo: an approach

To assess the adverse effect of a substance on the developing embryo, animal experimentation is employed. Approximately half of the tested compounds are found to cause detrimental skeletal damage. Therefore, an in vitro assay with the capacity to predict developmental osteotoxicity would be a highly valuable tool to reduce animal expenditure.Their capacity for self-renewal combined with their unique differentiation potential has led to embryonic stem cells (ESCs) becoming an attractive model system to study developmental processes.The fact that differentiating ESCs recapitulate early stages of mammalian development has made them a pivotal screening tool to identify potentially embryotoxic substances.The ZEBET laboratory uses both murine and primate embryonic stem cells as models to study osteogenic differentiation on a molecular level. Expression profiling of marker genes/proteinsis employed to identify and establish molecular endpoints indicative of skeletal damage during embryogenesis.A detailed description of the research strategy and preliminary results will be presented at the meeting

Human Multipotent Progenitors — A Promising Cell Model for Assessing Developmental Osteotoxicity In Vitro

To assess the potential adverse effects of substances on bone development in man, animalexperimentation is commonly employed. In vivo testing is labour- and cost-intensive andrequires a high number of laboratory animals. Up to now, there exists no validated alternativemethod to assess developmental bone toxicity in vitro. The derivation of multipotent progenitorswith mesenchymal characteristics from human embryonic stem cells (hES-MPs)constitutes one strategy in regenerative medicine, to work with a cell source that exhibits alow risk of tumour formation after transplantation. These cells display the typical morphologyof primary human mesenchymal stem cells and show a similar gene expression profile. Inaddition, they have a high proliferative activity and the capacity to differentiate into specialisedcell types of mesenchymal origin in vitro (adipocytes, chondrocytes, osteoblasts).Notably, their experimental use in Germany is exempt from regulatory approval enforced bythe German Stem Cell Act. Therefore, hES-MPs appear to be an attractive and promisinghuman-based cell model to screen for potential osteotoxic substances. Crucial stages duringosteogenesis involve the proliferation of progenitor cells, followed by their gradual differentiationinto functional osteoblasts, the maturation of the extracellular matrix (ECM) and, ultimately,the mineralisation of the ECM. Initial work to study the osteogenic differentiationprocess of hES-MPs has already been accomplished by another research group. Based on theirfindings, we further characterised the differentiation process regarding the influence of differentinducer cocktails, the delineation of the developmental stages and the expression of lineage-specific protein markers. A diverse range of biochemical and molecular biologicalmethods, e.g. cell viability and proliferation assays, colorimetric assays, cytochemical stainings,flow cytometry and western blotting, were employed to monitor the underlying molecularprocesses. Additionally, we performed initial experiments to investigate the sensitivity ofthe differentiating cells toward developmental toxicants. In summary, the hES-MP cell modelmight prove to be a valuable tool for assessing compound-mediated adversity on human bonedevelopment in vitro

Generation of ultrashort (<500 fs) wavelength tunable laser pulses by self-seeding and adiabatic soliton compression

Ultrashort, wavelength tunable laser pulses find an increasing range of quite different applications, like measurement techniques of ultrafast physical phenomena (e.g. electro-optic sampling), analysis of environmental data, and telecommunications. A simple way to generate wavelength tunable semiconductor lasers pulses having a width (FWHM) of a few picoseconds is self-seeding (SeSe) of a gain-switched Fabry-Perot (FP) laser diode and subsequent chirp compensation. This technique is particularly simple and low-cost. Neither highly sophisticated laser structures like, e.g. Tunable Twin Guide (TTG) or multi-section DBR lasers, nor anti-reflection coating of the facet, as required for mode-locking, is needed. Additional compression of SeSe pulses can be performed using nonlinear (soliton) effects in fibers. The potentially most promising method to generate ultrashort pulses of high quality is adiabatic pulse compression in dispersion decreasing fibers. In this contribution we demonstrated that it is indeed possible to generate femtosecond wavelength tunable pulses in such a way.</jats:p

Toxicological endpoints to assess developmental bone toxicity in vitro

To predict the toxic potential of industrial chemicals and pharmaceutical products on human bone development, rodent and non-rodent (e.g. rabbits) in vivo models are commonly used.The assessment of osteogenic toxicity in the embryo is integrated into the OECD testing guideline #414, which covers prenatal developmental toxicity. In experiments using rodents, onehalf of the litter (approximately 450 animals per test substance) is sacrificed for the examination of skeletal damage. In addition to the high number of laboratory animals required, these invivo studies are costly and time-consuming. Until today, there is no validated in vitro test to assess developmental bone toxicity.Therefore, current work at ZEBET in the frame of a joint project, funded by the German Ministry for Education and Research (BMBF), focuses on the development of a robust assay with the capacity to predict osteotoxicity in the embryo.Embryonic stem cells are pluripotent cells which can differentiate into a multitude of diverse cell types. Their capacity for unlimited self-renewal together with their ability to faithfullyrecapitulate early developmental programmes in the embryo in vitro makes them an extremely attractive model for embryonic toxicity studies.Crucial stages during osteogenesis involve the sequential expression of a tightly regulated set of molecular markers and the mineralisation of the extracellular matrix. We are currentlyassessing whether any of these markers can serve as a predictive endpoint to assess bone toxicity using a diverse range of molecular biological methods, e.g. real-time PCR, Western blot,flow cytometry, Fourier transform infrared spectroscopy (FTIR) and cytochemical staining. Mouse embryonic stem cells (line D3) are currently employed as a model system to study osteogenesis.As a next step this approach will be expanded to other cellular systems including stem cells from rhesus monkey, human iPS cells as well as human mesenchymal stem cells. The applicability of the different molecular markers and themorphological approach depending on the method is discussed regarding their potential as toxicological endpoint