Disentangling cellular proliferation and differentiation in the embryonic stem cell test, and its impact on the experimental protocol.
The mouse embryonic stem cell test (EST) was designed to predict embryotoxicity based on the inhibition of the differentiation of embryonic stem cells (ESC) into beating cardiomyocytes in combination with cytotoxicity data in monolayer ESC cultures and 3T3 cells. In the present study, we have tested a diverse group of chemicals in the EST, applying different exposure durations, in an attempt to discriminate between effects on proliferation and differentiation within the EST protocol. Chemicals tested were monobutyl phthalate (MBP), 6-aminonicotinamide (6-AN), 5-fluorouracil (5-FU) and 5-bromo-2'-deoxyuridine (BrdU). We showed that 5-FU and BrdU behaved principally different from MBP and 6-AN. 5-FU and BrdU specifically affected cell proliferation during the first three days of the EST protocol, as shown by EB size, protein concentration and cell cycle stage analysis. In addition, we studied the differentiation state of cells in the EST protocol with time to elucidate the transition of pluripotent ESC to more differentiated cell types. Analysis by flow cytometry of the pluripotency marker SSEA-1 in EST showed that although total SSEA-1 positive cells remained unchanged up to and including day 5, the signal intensity already decreased from day 3 onwards. Furthermore, RT-PCR data showed an upregulation of the mesodermal marker T at day 3, whereas the cardiac muscle marker Myh6 was upregulated from day 5 onwards. These findings confirm that proliferation and differentiation of ESC in the EST are highly intertwined processes. Based on these findings we suggest an amended EST protocol which could more clearly discriminate between proliferation and differentiation effects of chemicals within the same EST differentiation protocol. This proposal includes a cytotoxicity assessment in EB at day 3 of the EST after day 0-3 exposure, and cardiac muscle foci counts after exposure from day 3-10 in the EST.
van Dartel
Dorien A M
DA
Laboratory for Health Protection Research, National Institute of Public Health and the Environment (RIVM), 3720 BA, Bilthoven, The Netherlands. dorien.van.dartel@rivm.nl
Zeijen
Nicole J L
NJ
de la Fonteyne
Liset J J
LJ
van Schooten
Frederik J
FJ
Piersma
Aldert H
AH
D000826:Animal Testing Alternatives; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000502:physiology*; D005434:Flow Cytometry; D007202:Indicators and Reagents / Q000378:metabolism; D016256:Lewis X Antigen / Q000378:metabolism; D051379:Mice; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000502:physiology*; D010078:Oxazines / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods*; D014966:Xanthenes / Q000378:metabolism; D015262:Xenobiotics / Q000633:toxicity
D007202:Indicators and Reagents; D016256:Lewis X Antigen; D010078:Oxazines; D014966:Xanthenes; D015262:Xenobiotics; C005843:resazurin