Embryonic stem cells as a model to study the effect of antineoplastic drugs.

Background: Embryonic stem cells represent a suitable model to study cardiotoxic effect of endogenous or exogenous substances. Indeed the cardiotoxic effect of antineoplastic therapy is a common hitch in treated patients and limits the efficacy of therapy. Recently has been proposed that these effects, being cardiomyocytes terminally differentiated, may be due to "extra-sensitive" population represented by resident progenitors of cardiomyocytes.This may also explain the relatively high and delayed incidence of cardiac complications in patients treated for childhood cancers. However, the sensitivity of stem cells to antineoplastic drugs has not been investigated. We aimed to study the effect of antineoplastic drugs (doxorubicin and imatinib) on commitment, differentiation and maturation of stem cells toward cardiomyocytes. Methods: Mouse ES (mES, CGR8) cells were propagated in medium and differentiated into cardiomyocytes via formation of embryoid body (EB). We evaluated the effect of imatinib (1 and 0.1 M) and doxorubicin (Doxo, 0.1, 0.5 and 1 M)at different stage of differentiation process. The effect of drugs on viability was studied by MTT test. The effect of drugs on function were evaluated by measuring the percentage of beating EBs. To get information on the molecular mechanisms underlying or associated with the functional results, we used quantitative RT-PCR and we investigate the expression of transcription factor involved in cardiac differentiation (GATA-4, Nkx2.5, MEF2C). Results: Doxo added at first day of differentiation process does not allow EB formation. Thus, we added Doxo at EBs already formed, by exposing EBsfor 48 hours,starting from day 2 of EB formation.MTT demonstrate that Doxo has a very high toxicity and hinders EB formation, cell growth, and expression of cardiac specific markers. Surprisingly, when added to beating EBs, Doxo, does not influence the contractility. On the contrary, imatinibdoes not shows any toxic effect on EB formation, cell growth and contractility. Conclusion: Antineoplastic drugs exert different effects on cardiac commitment and differentiation of mES. Moreover, the window for Doxo toxicity seems to be restricted to the early stages of cell commitment, namely EB formation. Thus, it seems important to verify the effects of Doxo on cell aggregation and proliferation as well as expression of early mesodermic transcription factors.