| Abstract |
The assessment of adverse effects of substances on human embryonic bone formation is an integral part of regulatory in vivo studies that cover prenatal developmental toxicity. One of the most promising approaches to reduce 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. Nondifferentiating 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 EC50 values of 0.27, 3.2, and 0.0028?mM, respectively) at subtoxic concentrations (mean EC50 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. The assessment of adverse effects of substances on human embryonic bone formation is an integral part of regulatory in vivo studies that cover prenatal developmental toxicity. One of the most promising approaches to reduce 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. Nondifferentiating 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 EC50 values of 0.27, 3.2, and 0.0028?mM, respectively) at subtoxic concentrations (mean EC50 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.
|
