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  • In summary our work is an important step to

    2018-10-22

    In summary, our work is an important step to understand and to control phosphorylation dynamics in human pluripotent stem cell maintenance and differentiation. This is the first report that provides a detailed analysis of MAPKs regulation in hESC in response to a small molecule inhibitor. We have shown that a drastic decrease in phosphorylation of the three predominant MAPKs – p38, JNK and ERK – is induced upon EB formation. Secondly, in early EBs, addition of 5μM SB203580 resulted in further reduction of p38 phosphorylation but a moderate increase of JNK phosphorylation, which subsequently lead to increased cardiomyogenesis. Since p38 and HSP27 phosphorylation were essentially undetectable already at 5μM SB203580, higher concentrations apparently did not further affect the p38 pathway. In contrast, dominant JNK activation and clear ERK inhibition were observed, which was accompanied by a reduction in cardiomyogenesis and loss of cell viability. These results highlight the importance of a delicate balance of MAPKs pathway regulation for lineage-specific differentiation in hESC and explain the divergent effects of the small molecule inhibitor within a relative narrow range of concentrations.
    Materials and methods
    Acknowledgments
    Introduction FGF2 was first characterized in 1984 as a TAPI-1 146 amino TAPI-1 protein isolated from the pituitary gland (Bohlen et al., 1984). As a member of the FGF gene family, it is translated to produce 5 isotypes including one low-molecular-weight (18-kDa) and four high-molecular-weight (22, 22.5, 24 and 34-kDa) molecules (Delrieu, 2000). It serves as a potent and multifunctional growth factor that primarily targets endothelial cells, smooth muscle cells, fibroblasts, epithelial cells, and neural cells (Bikfalvi et al., 1997). In contrast to other growth factors, 18-kDa FGF2 does not have a signal sequence for secretion, and its mechanism of secretion is still unclear (Friesel and Maciag, 1995). Proposals suggest that FGF2 is primarily released from damaged or dead cells (Mignatti and Rifkin, 1991), secreted through the conventional endoplasmic reticulum (ER)-Golgi apparatus and released by chemicals containing phorbol ester PMA (Wang and Xu, 1998). In 1993, Forough described a recombinant cDNA that codes for the secreted form of human FGF-1 from NIH3T3 cells, which was constructed by adding the signal sequence of FGF4 encoding 22 amino acids to the 5′ end of the human full-length FGF-1 cDNA that encodes 154 amino acids (Forough et al., 1993). Another group used the same 22 amino acids of FGF4 to construct a replication-defective adenovirus fording for modified hFGF2. A large quantity of FGF2 was detected in both cell lysate and cultured medium of Cos cells infected with recombinant adenovirus (Ueno et al., 1997). In addition, a novel recombinant 4sFGF2 was constructed by replacing nine residues from the amino terminus of FGF2 with eight amino acid residues of the FGF4 signal sequence (Sohn et al., 2001). hESCs are derived from the inner cell mass of blastocysts and routinely cultured on mouse embryonic fibroblast (MEF) feeder layers or in fibroblast-conditioned medium (Thomson et al., 1998). FGF2 is routinely used for hESC culture and promotes hESC self renewal without differentiation (Dvorak et al., 2005). Several mechanisms may be involved to mediate the action of FGF2 in maintaining hESCs in an undifferentiated state. Firstly, FGF2 can directly activate the mitogen-activated protein kinase (MAPK) pathway in hESCs (Li et al., 2007), while indirectly activating transforming growth factor β1 (TGFβ1) (Greber et al., 2007) and activin A (Eiselleova et al., 2008). Secondly, the bone morphogenetic protein (BMP) antagonist noggin synergizes with FGF2 to repress BMP signals and sustains proliferation of hESCs in an undifferentiated state (Xu et al., 2005b). Thirdly, FGF2 contributes to the maintenance of hESCs by upregulating c-fos gene expression, one of the downstream targets of the MEK1/ERK (extracellular signal-regulated kinase) pathway (Kang et al., 2005). In addition, a recent study suggested that Nanog expression is enhanced by extrinsic FGF2 signaling (Xu et al., 2008; Yu et al., 2011). Finally, FGF2 binding to the surface of hESCs to regulate their proliferation is enhanced in the presence of MEF-secreted heparin sulfate proteoglycans in conditioned medium (Levenstein et al., 2008).