Monolayer cultivation of osteoprogenitors shortens duration of the embryonic stem cell test while reliably predicting developmental osteotoxicity.

Osteotoxic compounds administered during pregnancy can initiate skeletal congenital anomalies in the embryo. In vitro, developmental osteotoxicity of a compound can be predicted with the embryonic stem cell test (EST), the only in vitro embryotoxicity model identified to date that entirely abrogates the use of animals. Although the previously identified endpoint osteocalcin mRNA expression robustly predicts developmental osteotoxicity, it can only be assayed after 5 weeks of in vitro culture with existing embryoid body (EB)-based differentiation protocols. Therefore, the goal of this study was to characterize novel earlier endpoints of developmental osteotoxicity for the EST. The currently used EB-based differentiation protocol was modified so that a monolayer culture of pre-differentiated cells was inoculated. The expression profile of five bone-specific mRNAs, including osteocalcin, over the course of 30 differentiation days suggested an acceleration of pre-osteoblast specification in the monolayer over the EB-based protocol. Similarly, calcification was already visible after 14 days of culture in monolayer cultures. Employing image and absorption-based techniques to measure the degree of mineralization in these cells after compound treatment, the three compounds Penicillin G, 5-fluorouracil (5-FU) and all-trans retinoic acid (RA) were then tested after 14 days in monolayer cultures and compared to embryoid body-based differentiations at day 30. By modifying the culture the three test substances were classified correctly into non- or strong osteotoxic. Moreover, we were successful in shortening the assay duration from 30 to 14 days.