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.