br Experimental Procedures br Acknowledgments We would like

Experimental Procedures
Acknowledgments We would like to thank Dr. J.C. Louis (Amgen) for the CG4 cell line (Louis et al., 1992), Dr. J. Trotter (University of Mainz) for the Oli-neu cell line (Jung et al., 1995), J. Hightower for artwork, and Dr. M. Götz and members of the O.H. lab for valuable discussions. M.G.R. is a Howard Hughes Medical Institute Investigator. This study was supported by NIH grants (to M.G.R.), a Scientist Development Grant from the American Heart Association (to K.J.), and grants from the Swedish Brain Foundation (Hjärnfonden), David and Astrid Hagélen Foundation, SSMF (Svenska Sällskapet för Medicinsk Forskning), the Marie Curie Integration Grant, Seventh Framework Programme, European Union (to G.C.-B.), the Swedish Research Council (VR-MH), Karolinska Institutet Research Foundations, the Swedish Society of Medicine (SLS) (to G.C.-B. and O.H.), the Swedish Cancer Society (CF), the KI Cancer Network, the Swedish Foundation for Strategic Research (SSF), and the Swedish Childhood Cancer Foundation (BCF) (to O.H.).
Introduction Cell therapies to reverse muscle atrophy and to strengthen skeletal muscle would greatly enhance and extend the lives of patients with muscle wasting conditions due to diseases and/or aging. Embryonic stem igf1r inhibitor (ESCs) have unlimited proliferation potential, and no need for locating a suitable immunotype-matched donor as with adult-derived stem cells (Araki et al., 2013). However, a major obstacle in the development of ESC-based therapies targeting muscle has been the generation of a homogeneous myogenic population from in vitro differentiation, thus requiring optimization to enrich for muscle lineage cells. Several studies have validated the potential of mouse and human ESCs (mESCs and hESCs, respectively) and induced pluripotent stem cells (iPSCs), in skeletal muscle therapy (Barberi et al., 2007; Chang et al., 2009; Darabi et al., 2008, 2011a, 2011b, 2012; Sakurai et al., 2008). Cells were differentiated into paraxial mesoderm-like muscle progenitors, either by a standard serum-based embryoid body (EB) differentiation protocol (Chang et al., 2009; Sakurai et al., 2008) or by transient expression of PAX3 or PAX7 (Darabi et al., 2008, 2011a, 2012). These in vitro derived progenitors were able to engraft into adult myofibers of mice, replenish the muscle stem cell (satellite cell) niche, and enhance muscle contractile function (Chang et al., 2009; Darabi et al., 2008, 2011a, 2012; Sakurai et al., 2008). Despite promising results, these protocols are not appropriate for the generation of muscle progenitor cells (MPC) for clinical applications due to the inefficiency of differentiation and the use of viral vectors and potential insertional mutations (Thomas et al., 2003). Previous studies from our lab have used a serum-containing EB-induced differentiation supplemented with low levels of retinoic acid (RA) to enhance myogenesis from mouse (Kennedy et al., 2009) and human (Ryan et al., 2012) ESCs. However, serum-containing EB-differentiation of hESCs produced relatively low yields of skeletal muscle (<5%) and is undefined (Al Madhoun et al., 2011; Kennedy et al., 2009; Ryan et al., 2012). In contrast, directed differentiation uses knowledge of embryogenesis to recreate embryonic conditions in vitro using combinations of signaling molecules, to support the differentiation into one lineage (Murry and Keller, 2008). Applying the serum-free directed differentiation approach should greatly improve the efficiency of hESC-derived myogenesis for molecular analysis and for future use in cell therapies. Wnt signaling is critically important for the development of the primitive streak and paraxial mesoderm (Liu et al., 1999), marked by the T and MSGN1 or TBX6 genes, respectively, and in the formation of posterior somites and the tail bud (Takada et al., 1994), marked by the transcription factors PAX3, MEOX1, and PAX7. In the canonical pathway (reviewed in Clevers, 2006), Wnt binds to Frizzled cell-surface receptors, initiating a signaling cascade that inhibits GSK3B, preventing B-CATENNIN (CTNNB1) degradation, and allowing CTNNB1 to accumulate and translocate into the nucleus. Nuclear CTNNB1 enhances transcription by interaction with T cell factors or lymphocyte enhancer factors (Clevers, 2006).