An automated and high-throughput-screening compatible pluripotent stem cell-based test platform for developmental and reproductive toxicity assessment of small molecule compounds

TY - JOUR

T1 - An automated and high-throughput-screening compatible pluripotent stem cell-based test platform for developmental and reproductive toxicity assessment of small molecule compounds

AU - Witt, Gesa

AU - Keminer, Oliver

AU - Leu, Jennifer

AU - Tandon, Rashmi

AU - Meiser, Ina

AU - Willing, Anne

AU - Winschel, Ingo

AU - Abt, Jana-Christin

AU - Brändl, Björn

AU - Sébastien, Isabelle

AU - Friese, Manuel A

AU - Müller, Franz-Josef

AU - Neubauer, Julia C

AU - Claussen, Carsten

AU - Zimmermann, Heiko

AU - Gribbon, Philip

AU - Pless, Ole

PY - 2021/4

Y1 - 2021/4

N2 - The embryonic stem cell test (EST) represents the only validated and accepted in vitro system for the detection and classification of compounds according to their developmental and reproductive teratogenic potency. The widespread implementation of the EST, however, in particular for routine application in pharmaceutical development, has not been achieved so far. Several drawbacks still limit the high-throughput screening of potential drug candidates in this format: The long assay period, the use of non-homogeneous viability assays, the low throughput analysis of marker protein expression and the compatibility of the assay procedures to automation. We have therefore introduced several advancements into the EST workflow: A reduction of the assay period, an introduction of homogeneous viability assays, and a straightforward analysis of marker proteins by flow cytometry and high content imaging to assess the impact of small molecules on differentiation capacity. Most importantly, essential parts of the assay procedure have been adapted to lab automation in 96-well format, thus enabling the interrogation of several compounds in parallel. In addition, extensive investigations were performed to explore the predictive capacity of this next-generation EST, by testing a set of well-known embryotoxicants that encompasses the full range of chemical-inherent embryotoxic potencies possible. Due to these significant improvements, the augmented workflow provides a basis for a sensitive, more rapid, and reproducible high throughput screening compatible platform to predict in vivo developmental toxicity from in vitro data which paves the road towards application in an industrial setting. Graphical abstract •The embryonic stem cell test to predict teratogenicity was made automation-compatible. •Several key improvements to the assay procedure have been introduced to increase performance. •The workflow was adapted to human iPS cells and isogenic fibroblast donor cells.

AB - The embryonic stem cell test (EST) represents the only validated and accepted in vitro system for the detection and classification of compounds according to their developmental and reproductive teratogenic potency. The widespread implementation of the EST, however, in particular for routine application in pharmaceutical development, has not been achieved so far. Several drawbacks still limit the high-throughput screening of potential drug candidates in this format: The long assay period, the use of non-homogeneous viability assays, the low throughput analysis of marker protein expression and the compatibility of the assay procedures to automation. We have therefore introduced several advancements into the EST workflow: A reduction of the assay period, an introduction of homogeneous viability assays, and a straightforward analysis of marker proteins by flow cytometry and high content imaging to assess the impact of small molecules on differentiation capacity. Most importantly, essential parts of the assay procedure have been adapted to lab automation in 96-well format, thus enabling the interrogation of several compounds in parallel. In addition, extensive investigations were performed to explore the predictive capacity of this next-generation EST, by testing a set of well-known embryotoxicants that encompasses the full range of chemical-inherent embryotoxic potencies possible. Due to these significant improvements, the augmented workflow provides a basis for a sensitive, more rapid, and reproducible high throughput screening compatible platform to predict in vivo developmental toxicity from in vitro data which paves the road towards application in an industrial setting. Graphical abstract •The embryonic stem cell test to predict teratogenicity was made automation-compatible. •Several key improvements to the assay procedure have been introduced to increase performance. •The workflow was adapted to human iPS cells and isogenic fibroblast donor cells.

U2 - 10.1007/s10565-020-09538-0

DO - 10.1007/s10565-020-09538-0

M3 - SCORING: Journal article

C2 - 32564278

VL - 37

SP - 229

EP - 243

JO - CELL BIOL TOXICOL

JF - CELL BIOL TOXICOL

SN - 0742-2091

IS - 2

ER -