Human embryonic stem cells (hESCs) are a promising source for cell therapy in degenerative diseases. A key step in establishing the medical potential of hESCs is the development of techniques for the conversion of hESCs into tissue-restricted precursors suitable for transplantation. We recently described the derivation of multipotent mesenchymal precursors from hESCs. Nevertheless, our previous study was limited by the requirement for mouse feeders and the lack of in vivo data. Here we report a stroma-free induction system for deriving mesenchymal precursors. Selective culture conditions and fluorescence-activated cell sorting (FACS)-mediated purification yielded multipotent mesenchymal precursors and skeletal myoblasts. Skeletal muscle cells undergo in vitro maturation resulting in myotube formation and spontaneous twitching. We found that hESC-derived skeletal myoblasts were viable after transplantation into the tibialis anterior muscle of SCID/Beige mice, as assessed by bioluminescence imaging. Lack of teratoma formation and evidence of long-term myoblast engraftment suggests considerable potential for future therapeutic applications.

SUMMARY Cranial placodes are embryonic structures essential for sensory and endocrine organ development. Human placode development has remained largely inaccessible despite the serious medical conditions caused by the dysfunction of placode-derived tissues. Here, we demonstrate the efficient derivation of cranial placodes from human pluripotent stem cells. Timed removal of the BMP inhibitor Noggin, a component of the dual-SMAD inhibition strategy of neural induction, triggers placode induction at the expense of CNS fates. Concomitant inhibition of FGF signaling disrupts placode derivation and induces surface ectoderm. Further fate specification at the pre-placode stage enables the selective generation of placode-derived trigeminal ganglia capable of in vivo engraftment, mature lens fibers and anterior pituitary hormone-producing cells that upon transplantation produce human GH and ACTH in vivo. Our results establish a powerful experimental platform to study human cranial placode development and set the stage for the development of human cell-based therapies in sensory and endocrine disease.

AR is necessary for AR nuclear export and is dominant over the NLS in the DNAbinding domain and hinge region in the absence of hormone. Our findings suggest that androgen can regulate NES AR and, subsequently, the NLS of the AR, providing a mechanism by which androgen regulates AR nuclear/ cytoplasmic shuttling. Estrogen receptor ␣ and mineralocorticoid receptor also contain functional NES, suggesting that this ligand-regulated NES is conserved among steroid receptors. Androgen receptor (AR)1 is a member of the steroid receptor superfamily that regulates gene expression in a ligand-dependent manner (1, 2). AR is necessary for male accessory organ development and is involved in prostate cancer progression. The human AR contains 918 amino acids and consists of the N-terminal transactivation domain (1-555), DNA-binding domain (DBD) (556 -623), hinge region (624 -665), and C-terminal ligand-binding domain (LBD) (666 -918) (3). The DBD, hinge region, and LBD of AR share a high degree of homology with other steroid receptors. Intracellular localization of AR and other steroid receptors such as glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) is ligand-dependent. The limit for passive diffusion across the nuclear pore complex has been suggested to fall within the range of 20 -40 kDa (4). Larger proteins such as AR, which is ϳ100 kDa in size, must be actively transported through the nuclear pore complex to enter or leave the nucleus. AR is localized to the cytoplasm in the absence of ligand and translocates into the nucleus in the presence of ligand (5-7). Likewise, nuclear AR can be exported to the cytoplasm upon ligand withdrawal (8). These observations indicate that AR contains both a nuclear localization signal (NLS) and nuclear export signal (NES) and that their activities are regulated by androgen either directly or indirectly. Because nuclear localization is necessary for steroid receptors to transactivate their target genes, the ligand-dependent nuclear import/export represents a critical mechanism by which steroids regulate the activity of their cognate receptors.The signal involved in the transport of steroid receptors from the cytoplasm to the nucleus has been studied extensively. Mutagenesis studies of the human AR have defined a bipartite NLS in the DBD and hinge region at amino acids 617-633 (9). This NLS is composed of two clusters of basic amino acids (underlined) separated by 10 amino acid residues: RKCY-EAGMTLGARKLKK. Similar NLS were found in other steroid receptors. In addition to the NLS in the DBD and hinge region, Picard and Yamamoto (10) demonstrated the existence of a ligand-dependent NLS present in the LBD of GR. When fused to ␤-galactosidase or to N-terminal fragments of the GR lacking the NLS, the LBD of GR conferred ligand-dependent nuclear localization to the fusion proteins. Similarly, a ligand-dependent NLS also exists in the LBD of AR, which is capable of inducing nuclear import in the absence of the NLS in the DBD and hinge region (11,12).Studies on sequences responsible...

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