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  • In their letter Sadek et

    2018-10-24

    In their letter, Sadek et al. refer to other studies showing robust and reproducible regeneration. However, none of the studies published to date (Haubner et al., 2012; Heallen et al., 2013; Jesty et al., 2012; Naqvi et al., 2014; Strungs et al., 2013) actually performed apex resection on postnatal day 1 mice. As we have already stated in our paper (Andersen et al., 2014), we cannot exclude that cardiac regeneration may occur following other types of damage, such as myocardial infarction or cryoinjury (Haubner et al., 2012; Jesty et al., 2012; Naqvi et al., 2014; Strungs et al., 2013), that could leave a matrix beneficial for the repair process. Although it is possible that other surgery-related procedures not described in the published apex resection studies (Andersen et al., 2014; Mahmoud et al., 2014; Porrello et al., 2011) may actually diverge, we believe that the differences could be explained by how the amount of myocardium/fibrosis is assessed or interpreted. At P21, we observed that the scar in the heart located either posteriorly or anteriorly but seldom throughout the apex, which is in contrast to day 1–7 post surgery. We examindd more than 800 sections per heart and noted scarring in only 19.5%; hence, the damaged area could have been overlooked in other studies, including that of Porrello et al., 2011, where 140 sections per heart were examined (we apologize for stating “one heart” instead of “per heart” erroneously in our article [Andersen et al., 2014]).
    Acknowledgments
    Introduction Human embryonic stem dhpg (hESCs) and human induced pluripotent stem cells (hiPSCs), collectively known as human pluripotent stem cells (hPSCs), can potentially be differentiated into clinically useful cell types for in vitro disease modeling, drug screening, and cell replacement therapy. Given the explosion in diabetes and its complications worldwide, the directed differentiation of hPSCs into definitive endoderm (DE) and subsequently pancreatic cells is of immense interest (Teo et al., 2013a). In 2005, Novocell (now ViaCyte) reported the ability to derive > 80% of DE from hESCs with the use of 100 ng/ml Activin A (hereafter referred to as “Activin”) in the presence of 0.2%–2% fetal bovine serum (FBS; D’Amour et al., 2005). To complement Activin/Nodal signaling in inducing DE formation, Wnt and BMP signaling activators were then introduced (Table S1 available online). Developmentally, this mimics the complex Nodal, Wnt (Wnt3, Wnt3a and Wnt5a) and Bmp (Bmp4) signaling, which operate during gastrulation, primitive streak, and early DE formation in the mouse embryo (Arnold and Robertson, 2009; Lawson et al., 1999; Liu et al., 1999; Ohinata et al., 2009; Teo et al., 2011; Yamaguchi et al., 1999). The coactivation of Wnt and Activin/Nodal signaling (albeit in the presence of FBS) is commonly used as described by D’Amour et al. (2006). They reported that Activin and WNT3A (specifically 100 ng/ml Activin, 25 ng/ml WNT3A, and 0.2% FBS) can induce more than 80% of DE cells (D’Amour et al., 2006). Alternatively, activation of Wnt signaling via the inhibition of glycogen synthase kinase (GSK)-3 (specifically 100 ng/ml Activin or 500 nM IDE1, 3 μM CHIR99021 [GSK-3 inhibitor], and 2% FBS) has also been recently reported to induce 70%–80% of DE cells (Illing et al., 2013; Kunisada et al., 2012). In contrast, the work of Teo, Dunn (Phillips et al., 2007; Teo et al., 2012), and Vallier (Vallier et al., 2009) independently demonstrated that 10–50 ng/ml of BMP4 can synergistically act with Activin (in a defined medium without FBS) to induce more than 80% of DE cells. However, it is still uncertain how Wnt compares with BMP in cooperating with Activin/Nodal signaling in a chemically defined medium to induce DE formation. We sought to clarify and define the Activin-Wnt-BMP signaling relationship using hiPSCs that we derived (Teo et al., 2013b). Here, we report a head-to-head comparison of Wnt versus BMP in combination with Activin signaling in a chemically defined medium without FBS to induce DE formation. Unlike previous reports (25 ng/ml WNT3A plus FBS; Table S1), we observed that a high dose of WNT3A (100 ng/ml, without FBS) is required to maximally induce DE in the presence of 100 ng/ml Activin. The activation of Wnt signaling with the use of two independent GSK-3 inhibitors (CHIR99021 or 6-bromoindirubin-3′-oxime [BIO]; Sato et al., 2004) or BMP signaling with BMP4 can also maximally induce DE cells. Thus, both Wnt and BMP signaling can effectively cooperate with Activin signaling to induce DE formation.