Osteogenic Differentiation of Human Embryonic Stem Cell-Derived Mesenchymal Progenitor Cells as a Model for Assessing Developmental Bone Toxicity In Vitro

Abstract

The assessment of adverse effects of substances on human embryonic bone formation is an integral part of reg-ulatory in vivo studies that cover prenatal developmental toxicity. One of the most promising approaches to re-duce such in vivo testing is the application of stem cell-based in vitro methods. In the present study, we aimed to establish an in vitro system for developmental bone toxicity testing using human embryonic stem cell-derived mesenchymal progenitors (hES-MPs). Cell-based and biochemical assays as well as cytochemical stainings and flow cytometry confirmed the presence of a differentiation pattern characteristic for osteogenesis. Nondif-ferentiating hES-MPs highly expressed the early protein markers Runx2 and Dlx5, while alkaline phosphatase expression and activity strongly increased during differentiation. Moreover, immunofluorescence staining revealed the formation of a compact collagen-rich extracellular matrix, which quickly mineralized. Based on these results, we established toxicological endpoints. Cells were differentiated in the presence of test chemicals from day 1 to 15 and analyzed for osteogenic differentiation and cytotoxicity. Differentiation was assessed by applying an easy-to-use fluorescence-based mineralization assay, and cell viability was determined by resazurin reduction. Treatments with the developmental toxicants sodium valproate, boric acid, and warfarin revealed a specific effect on mineralization (with mean EC 50 values of 0.27, 3.2, and 0.0028 mM, respectively) at subtoxic concentrations (mean EC 50 values for cell viability were 7.8, > 8.3, and > 0.3 mM, respectively). A significantly stronger effect on mineralization in comparison to cell viability was observed with p values of p = 0.013, 0.0049, and 0.015, respectively. In contrast, the antineoplastic agent 5-fluorouracil and the nondevelopmental toxicants diphenhydramine, metoclopramide, and penicillin G had no significantly stronger impact on mineralization in comparison to cell viability with p values of p 0.46. In summary, the teratogenic potential for six of the seven test chemicals was correctly predicted with this assay.