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  • Taken together CHIR appears to be


    Taken together, CHIR99021 appears to be the preferred choice in combination with Activin (rank 1, Figure 4D) in generating DE cells based on its superior ability to induce mesendoderm as well as its cost effectiveness (∼2 cents/ml of media). BMP4 would be favored if not for its ∼20-fold greater cost (rank 2, Figure 4D), whereas BIO, although less expensive, exhibits cytotoxic effects (rank 3, Figure 4D) and WNT3A is both expensive and requires a high dose to be effective (rank 4, Figure 4D). In summary, we demonstrate that both Wnt and BMP can cooperate with Activin signaling to induce DE with comparable efficiencies in a chemically defined serum-free medium. DE generated from both Wnt+Activin and BMP+Activin signaling can give rise to derivatives such as pancreatic progenitor cells (Kunisada et al., 2012; Teo et al., 2012). Thus, DE made via these two differing signaling pathways is conceptually consistent and physiologically relevant. Future genome-wide comparisons could reveal interesting mechanistic insights specific to Wnt and BMP signaling that are relevant for DE specification.
    Experimental Procedures
    Introduction In most vertebrates, the process of myogenic differentiation entails the withdrawal of precursors from the cell cycle, followed by their fusion into myotubes. The multinucleate state is characterized by a permanent postmitotic arrest, which renders the myotubes unable to respond to proliferative cues (Pajalunga et al., 2008; Walsh and Perlman, 1997). In contrast, salamander myotubes remain responsive to such cues, being able to re-enter the find more info upon serum stimulation in culture (Tanaka et al., 1997) or after implantation within regenerating structures (Kumar et al., 2000). In salamander (Notophthalmus viridescens) A1 myotubes (Ferretti and Brockes, 1988), serum stimulation induces a reprogramming process that includes partial dedifferentiation, as suggested by the downregulation of the myogenic gene Myf5 (Imokawa et al., 2004), and re-entry into the cell cycle, which is also considered an aspect of dedifferentiation. The latter depends on the phosphorylation of Rb (Tanaka et al., 1997) and the downregulation of p53 activity (Yun et al., 2013). The serum component that triggers these responses is not a conventional growth factor but an as-yet-unidentified thrombin-activated serum component that acts as a mitogen for myotubes, but not for mononucleate precursors (Lööf et al., 2007; Straube et al., 2004; Tanaka et al., 1999). Even though mammalian myotube nuclei cannot be reprogrammed upon exposure to this factor (Lööf et al., 2007), they are able to re-enter the cell cycle after forming heterokaryons with salamander myotubes (Velloso et al., 2001). This suggests that even when the initial response may be different, part of the pathway leading to serum-mediated reprogramming is conserved. Both the identity of the serum factor and the signaling pathways driving the reversal of the differentiated state in regeneration-competent salamander cells remain unknown, although extensive efforts to identify the serum factor are find more info ongoing (Straube et al., 2004). In proliferating cells, the extracellular signal-regulated kinase (ERK) family of mitogen-activated protein kinases (MAPKs) plays a critical role in driving cell-cycle progression in a variety of cell types (Albeck et al., 2013; Cook and McCormick, 1996; Murphy et al., 2002; Weber et al., 1997; Yamamoto et al., 2006). In fibroblasts, sustained ERK activation is required for successful S phase progression by promoting the downregulation of antiproliferative genes during G1 phase and controlling the state of Rb phosphorylation (Yamamoto et al., 2006). Hence, it is possible that ERK activation plays a role during the reprogramming of differentiated salamander cells. Herein, we have tested this hypothesis using the salamander A1 cell line as a model for serum-induced reprogramming.