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Here, we report that MYC rescues early human cells undergoing reprogramming from a proliferation pause induced by OCT3/4, SOX2, and KLF4 (OSK). We identified ESRG as a marker of early reprogramming cells that is expressed as early as day 3 after OSK induction. On day 4, ESRG positive (+) cells converted to a TRA-1-60 (+) intermediate state. These early ESRG (+) or TRA-1-60 (+) cells showed a proliferation pause due to increased p16INK4A and p21 and decreased endogenous MYC caused by OSK. Exogenous MYC did not enhance the appearance of initial reprogramming cells but instead reactivated their proliferation and improved reprogramming efficiency. MYC increased expression of LIN41, which potently suppressed p21 post-transcriptionally. MYC suppressed p16 INK4A. These changes inactivated retinoblastoma protein (RB) and reactivated proliferation. The RB-regulated proliferation pause does not occur in immortalized fibroblasts, leading to high reprogramming efficiency even without exogenous MYC.

Reprogramming differentiated cells into induced pluripotent stem cells (iPSCs) promotes a broad array of cellular changes. Here we show that the let-7 family of microRNAs acts as an inhibitory influence on the reprogramming process through a regulatory pathway involving prodifferentiation factors, including EGR1. Inhibiting let-7 in human cells promotes reprogramming to a comparable extent to c-MYC when combined with OCT4, SOX2, and KLF4, and persistence of let-7 inhibits reprogramming. Inhibiting let-7 during reprogramming leads to an increase in the level of the let-7 target LIN-41/TRIM71, which in turn promotes reprogramming and is important for overcoming the let-7 barrier to reprogramming. Mechanistic studies revealed that LIN-41 regulates a broad array of differentiation genes, and more specifically, inhibits translation of EGR1 through binding its cognate mRNA. Together our findings outline a let-7-based pathway that counteracts the activity of reprogramming factors through promoting the expression of prodifferentiation genes.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2007.11.019 Byline: Kazutoshi Takahashi (1), Koji Tanabe (1), Mari Ohnuki (1), Megumi Narita (1)(2), Tomoko Ichisaka (1)(2), Kiichiro Tomoda (3), Shinya Yamanaka (1)(2)(3)(4) Abstract: Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells, capable of germline transmission, from mouse somatic cells by transduction of four defined transcription factors. Here, we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. Furthermore, these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts. Author Affiliation: (1) Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan (2) CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan (3) Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA (4) Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japan Article History: Received 29 October 2007; Revised 7 November 2007; Accepted 12 November 2007 Article Note: (miscellaneous) Published online: November 20, 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.01.042 Byline: Tadayoshi Watanabe (a)(b), Daisuke Saito (a), Koji Tanabe (b)(c), Rinako Suetsugu (b), Yukiko Nakaya (b), Shinichi Nakagawa (b)(c), Yoshiko Takahashi (a)(b) Keywords: Tet-on; Tet-off; In ovo electroporation; Somitogenesis; RhoA; cMeso-1; Pax2; NCAM; Cell condensation Abstract: The in ovo electroporation technique in chicken embryos has enabled investigators to uncover the functions of numerous developmental genes. In this technique, the ubiquitous promoter, CAGGS (CMV base), has often been used for overexpression experiments. However, if a given gene plays a role in multiple steps of development and if overexpression of this gene causes fatal consequences at the time of electroporation, its roles in later steps of development would be overlooked. Thus, a technique with which expression of an electroporated DNA can be controlled in a stage-specific manner needs to be formulated. Here we show for the first time that the tetracycline-controlled expression method, 'tet-on' and 'tet-off', works efficiently to regulate gene expression in electroporated chicken embryos. We demonstrate that the onset or termination of expression of an electroporated DNA can be precisely controlled by timing the administration of tetracycline into an egg. Furthermore, with this technique we have revealed previously unknown roles of RhoA, cMeso-1 and Pax2 in early somitogenesis. In particular, cMeso-1 appears to be involved in cell condensation of a newly forming somite by regulating Pax2 and NCAM expression. Thus, the novel molecular technique in chickens proposed in this study provides a useful tool to investigate stage-specific roles of developmental genes. Author Affiliation: (a) Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara, 630-0192, Japan (b) Center for Developmental Biology, RIKEN, 2-2-3 Minatojima Minamimachi, Chuo-ku, Kobe 650-0047, Japan (c) Nakagawa Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan Article History: Received 21 September 2006; Revised 27 January 2007; Accepted 29 January 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.01.043 Byline: Yuki Sato (a)(b), Toshiharu Kasai (b), Shinichi Nakagawa (b)(c), Koji Tanabe (b)(c), Tadayoshi Watanabe (a)(b), Koichi Kawakami (d), Yoshiko Takahashi (a)(b) Keywords: Transposon; In ovo electroporation; Chicken; Genomic integration; Tetracycline Abstract: The in ovo electroporation in chicken embryos has widely been used as a powerful tool to study roles of genes during embryogenesis. However, the conventional electroporation technique fails to retain the expression of transgenes for more than several days because transgenes are not integrated into the genome. To overcome this shortcoming, we have developed a transposon-mediated gene transfer, a novel technique in chicken manipulations. It was previously reported that the transposon Tol2, originally found in medaka fish, facilitates an integration of a transgene into the genome when co-acting with Tol2 transposase. In this study, we co-electroporated a plasmid containing a CAGGS-EGFP cassette cloned in the Tol2 construct along with a transposase-encoding plasmid into early presomitic mesoderm or optic vesicles of chicken embryos. This resulted in persistent expression of EGFP at least until embryonic day 8 (E8) and E12 in somite-derived tissues and developing retina, respectively. The integration of the transgene was confirmed by genomic Southern blotting using chicken cultured cells. We further combined this transposon-mediated gene transfer with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late organogenesis, for which chickens are most suitable model animals. Author Affiliation: (a) Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara, 630-0192, Japan (b) Center for Developmental Biology, RIKEN, 2-2-3 Minatojima Minamimachi, Chuo-ku, Kobe 650-0047, Japan (c) Nakagawa Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan (d) Division of Molecular and Developmental Biology, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan Article History: Received 21 September 2006; Revised 27 January 2007; Accepted 29 January 2007

Collagen VI is a key component of muscle basement membranes, and genetic variants can cause monogenic muscular dystrophies. Conversely, human genetic studies recently implicated collagen VI in central nervous system function, with variants causing the movement disorder dystonia. To elucidate the neurophysiological role of collagen VI, we generated mice with a truncation of the dystoniarelated collagen a3 VI (COL6A3) C-terminal domain (CTD). These Col6a3CTT mice showed a recessive dystonia-like phenotype in both sexes. We found that COL6A3 interacts with the cannabinoid receptor 1 (CB1R) complex in a CTD-dependent manner. Col6a3CTT mice of both sexes have impaired homeostasis of excitatory input to the basal pontine nuclei (BPN), a motor control hub with dense COL6A3 expression, consistent with deficient endocannabinoid (eCB) signaling. Aberrant synaptic input in the BPN was normalized by a CB1R agonist, and motor performance in Col6a3CTT mice of both sexes was improved by CB1R agonist treatment. Our findings identify a readily therapeutically addressable synaptic mechanism for motor control. [ABSTRACT FROM AUTHOR]