Detection of tissue-specific effects by methotrexate on differentiating mouse embryonic stem cells.
BACKGROUND
Pluripotent embryonic stem (ES) cells offer a unique possibility to monitor the differentiation of several cell types in vitro. This study attempts to identify marker genes during in vitro cell differentiation of murine ES cells and allow a prediction of chemical effects on cell differentiation of specific target tissues. The study focused on the expression pattern of key genes involved in cardiomyocyte and osteoblast differentiation: Oct-4, Brachyury, Nkx2.5, alpha myosin heavy chain, Cbfa1, and Osteocalcin.


METHODS
Methotrexate was selected due to its well-characterized teratogenic effects. Several in vivo studies have demonstrated the specific interactions of methotrexate with bone formation whereas the cardiovascular system is not specifically affected after exposure to low concentration. The capability of murine ES cells to differentiate in vitro into cardiomyocytes as well as into osteoblasts have been used to demonstrate the target cell specificity in vitro, at non-cytotoxic concentration.


RESULTS
Exposure of differentiating ES cells did not result in any gene profile modification of the selected cardiomyocyte specific genes, whereas the expression of osteoblast specific key genes, Cbfa1 and Osteocalcin, decreased. At the latter stages of skeletal differentiation we observed a 30% decrease in gene expression for Cbfa1 and a 60% decrease for Osteocalcin, with reference to the control. Early marker genes for undifferentiated cells and mesodermal cells were not modified after methotrexate treatment.


CONCLUSIONS
These results show the possibility to integrate specific in vitro tests for teratogenicity in a test strategy for developmental toxicity.
Pellizzer
Cristian
C
European Centre for the Validation of Alternative Methods, Institute for Health and Consumer Protection, Joint Research Centre, Ispra, Italy.
Bello
Ezia
E
Adler
Sarah
S
Hartung
Thomas
T
Bremer
Susanne
S
D000818:Animals; D002113:Calcification, Physiologic / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D004622:Embryo, Mammalian / Q000166:cytology*; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000235:genetics; D006321:Heart / Q000254:growth & development; D008727:Methotrexate / Q000633:toxicity*; D051379:Mice; D024510:Muscle Development / Q000187:drug effects / Q000235:genetics; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000235:genetics; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D013234:Stem Cells / Q000187:drug effects*; D014157:Transcription Factors / Q000235:genetics
D012333:RNA, Messenger; D014157:Transcription Factors; D015675:Osteocalcin; D018995:Myosin Heavy Chains; D008727:Methotrexate