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Trends in improving the embryonic stem cell test (EST): an overview
Roland Buesen1, Anke Visan, Elke Genschow
1Center for Documentation and Evaluation of Alternative Methods to Animal Experiments at the Federal Institute for Risk Assessment, D-Berlin, Germany.
ALTEX
|February 21, 2004
Summary
The embryonic stem cell test (EST) offers a reliable in vitro method for assessing chemical embryotoxicity. Enhanced molecular endpoints, like flow cytometry, improve prediction accuracy and enable high-throughput screening for developmental toxicity.
Area of Science:
- Developmental toxicology
- In vitro toxicology
- Stem cell biology
Background:
- The embryonic stem cell test (EST) is an established in vitro assay for evaluating teratogenic and embryotoxic potential.
- It relies on the differentiation of murine embryonic stem cells (ES cells) into contracting cardiomyocytes as a toxicological endpoint.
- Previous validation studies demonstrated its reliability comparable to animal-based in vitro tests.
Purpose of the Study:
- To improve the embryonic stem cell test (EST) by incorporating molecular endpoints for enhanced accuracy.
- To evaluate the utility of intracellular flow cytometry for quantifying cardiac-specific protein expression as a measure of embryotoxicity.
- To explore the potential of flow cytometry for high-throughput screening (HTS) applications in developmental toxicology.
Main Methods:
- Cultured murine ES cells (D3) were exposed to chemicals with varying embryotoxic potential.
- Differentiation into cardiomyocytes was assessed using both traditional microscopic analysis of beating areas and intracellular flow cytometry (FACS) for cardiac-specific protein expression.
- New protocols were developed to stimulate differentiation into neural, endothelial, chondrocyte, and osteoblast lineages, with corresponding molecular endpoints.
Main Results:
- Flow cytometry analysis of cardiac-specific protein expression provided a more objective and reliable endpoint for predicting chemical embryotoxicity compared to microscopic beating area assessment.
- The developed molecular endpoints successfully detected specific differentiation pathways.
- New prediction models (PMs) were created utilizing single EST endpoints, enhancing predictive capabilities.
Conclusions:
- The improved EST, incorporating molecular endpoints and flow cytometry, offers a more objective and potentially higher-throughput method for assessing chemical embryotoxicity.
- The integration of diverse differentiation protocols and molecular endpoints broadens the scope of the EST to detect effects on various cell types.
- These advancements contribute to more accurate and efficient in vitro safety assessments for drugs and chemicals, reducing reliance on animal testing.