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Co-culture with neonatal cardiomyocytes enhances the proliferation of iPSC-derived cardiomyocytes via FAK/JNK signaling.

ABSTRACT: We previously reported that the pluripotent stem cells can differentiate into cardiomyocytes (CMs) by co-culture with neonatal CMs (NCMs) in vitro. However, the involving mechanism is not clear.Mouse induced pluripotent stem cells (iPSCs) were cultured in hanging drops to form embryoid bodies (EBs) and to induce myocardial differentiation. Co-culture of EBs and NCMs was established in a transwell insert system, while EBs grown alone in the wells were used as controls.Co-culture with NCMs markedly increased the generation of functional CMs from iPSCs. The focal adhesion kinase (FAK) phosphorylation, and c-Jun N-terminal kinase (JNK) phosphorylation in co-culture were higher than that in EBs grown alone. Treating FAK small interfering RNA (FAK siRNA) or specific inhibitor for JNK (SP600125) to iPSCs significantly reduced the phosphorylation of JNK and the expressions of Mef2c and Bcl-2. The expressions of cTnT and MLC-2V were also decreased. Our results revealed that co-culture with NCMs significantly enhance the differentiation ability of iPSCs by increasing Mef2c and Bcl-2 expressions concomitantly with a marked augment on cell proliferation through JNK signaling pathways.These findings indicated that co-culture of EBs with NCMs induces genes expressed in a mature pattern and stimulates the proliferation of iPSC-derived CMs (iPS-CMs) by activating FAK/JNK signaling.

SUBMITTER: Ou D

PROVIDER: S-EPMC4855360 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Co-culture with neonatal cardiomyocytes enhances the proliferation of iPSC-derived cardiomyocytes via FAK/JNK signaling.

Ou Dongbo D Wang Qi Q Huang Yanjin Y Zeng Di D Wei Ting T Ding Lu L Li Xiaoli X Zheng Qiangsun Q Jin Yan Y

BMC developmental biology 20160504

<h4>Background</h4>We previously reported that the pluripotent stem cells can differentiate into cardiomyocytes (CMs) by co-culture with neonatal CMs (NCMs) in vitro. However, the involving mechanism is not clear.<h4>Methods</h4>Mouse induced pluripotent stem cells (iPSCs) were cultured in hanging drops to form embryoid bodies (EBs) and to induce myocardial differentiation. Co-culture of EBs and NCMs was established in a transwell insert system, while EBs grown alone in the wells were used as co ...[more]

PMID: 27141946

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Project description:Because mammalian cardiomyocytes largely cease to proliferate immediately after birth, the regenerative activity of the heart is limited. To date, much effort has been made to clarify the regulatory mechanism of cardiomyocyte proliferation because the amplification of cardiomyocytes could be a promising strategy for heart regenerative therapy. Recently, it was reported that the inhibition of glycogen synthase kinase (GSK)-3 promotes the proliferation of neonatal rat cardiomyocytes (NRCMs) and human iPS cell-derived cardiomyocytes (hiPSC-CMs). Additionally, Yes-associated protein (YAP) induces cardiomyocyte proliferation. The purpose of this study was to address the importance of YAP activity in cardiomyocyte proliferation induced by GSK-3 inhibitors (GSK-3Is) to develop a novel strategy for cardiomyocyte amplification. Immunofluorescent microscopic analysis using an anti-Ki-67 antibody demonstrated that the treatment of NRCMs with GSK-3Is, such as BIO and CHIR99021, increased the ratio of proliferative cardiomyocytes. YAP was localized in the nuclei of more than 95% of cardiomyocytes, either in the presence or absence of GSK-3Is, indicating that YAP was endogenously activated. GSK-3Is increased the expression of β-catenin and promoted its translocation into the nucleus without influencing YAP activity. The knockdown of YAP using siRNA or pharmacological inhibition of YAP using verteporfin or CIL56 dramatically reduced GSK-3I-induced cardiomyocyte proliferation without suppressing β-catenin activation. Interestingly, the inhibition of GSK-3 also induced the proliferation of hiPSC-CMs under sparse culture conditions, where YAP was constitutively activated. In contrast, under dense culture conditions, in which YAP activity was suppressed, the proliferative effects of GSK-3Is on hiPSC-CMs were not detected. Importantly, the activation of YAP by the knockdown of α-catenin restored the proproliferative activity of GSK-3Is. Collectively, YAP activation potentiates the GSK-3I-induced proliferation of cardiomyocytes. The blockade of GSK-3 in combination with YAP activation resulted in remarkable amplification of cardiomyocytes.

Dataset Information

Co-culture with neonatal cardiomyocytes enhances the proliferation of iPSC-derived cardiomyocytes via FAK/JNK signaling.

Publications

Co-culture with neonatal cardiomyocytes enhances the proliferation of iPSC-derived cardiomyocytes via FAK/JNK signaling.

Similar Datasets

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