Early embryonic sensitivity to cyclophosphamide in cardiac differentiation from human embryonic stem cells

Authors

  • Ming-Xia Zhu,

    1. Occupational Disease Research Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, Peoples Republic of China
    2. Stem Cell Research Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, Peoples Republic of China
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  • Jin-Yuan Zhao,

    Corresponding author
    1. Occupational Disease Research Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, Peoples Republic of China
    2. Stem Cell Research Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, Peoples Republic of China
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  • Gui-An Chen,

    Corresponding author
    1. Stem Cell Research Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, Peoples Republic of China
    2. Reproductive Medical Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, Peoples Republic of China
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  • Li Guan

    1. Occupational Disease Research Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, Peoples Republic of China
    2. Stem Cell Research Center, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing 100191, Peoples Republic of China
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Correspondence may be addressed to either of these authors (email zhaojinyuan.bj@gmail.com or chenguian2008@bjmu.edu.cn).

Abstract

hESCs (human embryonic stem cells) can differentiate into tissue derivatives of all three germ layers in vitro and mimic the development of the embryo in vivo. In this study, we have investigated the potential of an hESC-based assay for the detection of toxicity to cardiac differentiation in embryonic development. First of all, we developed the protocol of cardiac induction from hESCs according to our previous work and distinguished cardiac precursor cells and late mature cardiomyocytes from differentiated cells, demonstrated by the Q-PCR (quantitative real-time PCR), immunocytochemistry and flow cytometry analysis. In order to test whether CPA (cyclophosphamide) induces developmental and cellular toxicity in the human embryo, we exposed the differentiating cells from hESCs to CPA (a well-known proteratogen) at different stages. We have found that a high concentration of CPA could inhibit cardiac differentiation of hESCs. Two separate exposure intervals were used to determine the effects of CPA on cardiac precursor cells and late mature cardiomyocytes respectively. The cardiac precursor cells were sensitive to CPA in non-cytotoxic concentrations for the expression of the cardiac-specific mRNA markers Nkx2.5 (NK2 transcription factor related, locus 5), GATA-4 (GATA binding protein 4 transcription factor) and TNNT2 (troponin T type 2). Non-cytotoxic CPA concentrations did not affect the mRNA markers' expression in late mature cardiomyocytes, indicating that cardiac precursors were more sensitive to CPA than late cardiomyocytes in cardiogenesis. We set up the in vitro developmental toxicity test model so as to reduce the number of test animals and expenses without compromising the safety of consumers and patients. Furthermore, such in vitro methods may be possibly suited to test a large number of chemicals than the classical employed in vivo tests.

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