The use of quantitative image analysis in the assessment of in vitro embryotoxicity endpoints based on a novel embryonic stem cell clone with endoderm-related GFP expression.
The capacity of pluripotent embryonic stem cells (ESC) to differentiate in vitro into various tissues provides the opportunity to develop an in vitro assay for investigating mechanisms of developmental toxicity. ESC clones carrying tissue specific reporter gene constructs are currently being developed. The clones should allow the quantification of the effects of chemicals on the development of germ layers and main target tissues. We report the establishment of the alpha-fetoprotein_GFP/D3 reporter gene clone: alpha-fetoprotein (AFP) enhancers and the homologous promoter regulate green fluorescent protein (GFP) expression in cells of the D3-ESC clone. AFP was used as a marker for endodermal cells. Differentiation of this clone via embryoid bodies (EBs, spheroids of cells) leads to green fluorescence on the surfaces of EBs. AFP- related GFP expression was confirmed. An easy and quick image analysis-based endpoint measurement was developed for quantifying low amounts of cells expressing GFP. As demonstrated with the embryotoxic chemical diphenylhydantoin, image analysis can be used to distinguish between a general effect on EB growth and a specific effect on the development of GFP-positive endodermal cells. Endoderm development was inhibited at a different dose than cardiomyocyte development.
ECVAM, Institute for Health and Consumer Protection, Joint Research Centre, European Commission, I-21020 Ispra (VA), Italy.
M
M
Paparella
E
E
Kolossov
B K
BK
Fleischmann
J
J
Hescheler
S
S
Bremer
D000826:Animal Testing Alternatives; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D002999:Clone Cells; D004305:Dose-Response Relationship, Drug; D004707:Endoderm / Q000187:drug effects / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000187:drug effects / Q000378:metabolism*; D018874:Spheroids, Cellular / Q000166:cytology / Q000187:drug effects; D015262:Xenobiotics / Q000633:toxicity; D000509:alpha-Fetoproteins / Q000235:genetics
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D015262:Xenobiotics; D000509:alpha-Fetoproteins; D049452:Green Fluorescent Proteins