Detection of tissue-specific effects by methotrexate on differentiating mouse embryonic stem cells

Cristian Pellizzer, Ezia Bello, Sarah Adler, Thomas Hartung, Susanne Bremer

Research output: Contribution to journalArticle

Abstract

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, α 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.

Original languageEnglish (US)
Pages (from-to)331-341
Number of pages11
JournalBirth Defects Research Part B - Developmental and Reproductive Toxicology
Volume71
Issue number5
DOIs
StatePublished - Oct 1 2004
Externally publishedYes

Keywords

  • Cardiac differentiation
  • Methotrexate
  • Mouse embryonic stem cells
  • RT-PCR
  • Skeletal differentiation
  • Teratogenicity

ASJC Scopus subject areas

  • Embryology
  • Toxicology
  • Developmental Biology
  • Health, Toxicology and Mutagenesis

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