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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]

SUMMARY There is a strong interest in onsite rapid analysis and monitoring of the activity of cells and biological samples. Such analyses contribute not only to the understanding of cell functions, but also to the improvement of point-of-care testing and quality-of-life. In promoting miniaturization and integration of sensing components, electrochemistry is advantageous. By coupling this technique with microfluidic techniques, additional functions that can never be realized with only independent sensors can be realized. In this review paper, we introduce our recent electrochemical devices, particularly focusing on analyses of cell functions and biological samples. [ABSTRACT FROM AUTHOR]

Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic β cells is important for understanding and treating diabetes. The pancreatic β cell line, MIN6, retains GSIS but gradually loses it in long-term culture. The MIN6 subclone, MIN6c4, exhibits well-regulated GSIS even after prolonged culture. We previously used DNA microarray analysis to compare gene expression in the parental MIN6 cells and MIN6c4 cells and identified several differentially regulated genes that may be involved in maintaining GSIS. Here we investigated the potential roles of six of these genes in GSIS: Tmem59l (Transmembrane protein 59 like), Scgn (Secretagogin), Gucy2c (Guanylate cyclase 2c), Slc29a4 (Solute carrier family 29, member 4), Cdhr1 (Cadherin-related family member 1), and Celsr2 (Cadherin EGF LAG seven-pass G-type receptor 2). These genes were knocked down in MIN6c4 cells using lentivirus vectors expressing gene-specific short hairpin RNAs (shRNAs), and the effects of the knockdown on insulin expression and secretion were analyzed. Suppression of Tmem59l, Scgn, and Gucy2c expression resulted in significantly decreased glucose- and/or KCl-stimulated insulin secretion from MIN6c4 cells, while the suppression of Slc29a4 expression resulted in increased insulin secretion. Tmem59l overexpression rescued the phenotype of the Tmem59l knockdown MIN6c4 cells, and immunostaining analysis indicated that the TMEM59L protein colocalized with insulin and GM130, a Golgi complex marker, in MIN6 cells. Collectively, our findings suggested that the proteins encoded by Tmem59l, Scgn, Gucy2c, and Slc29a4 play important roles in regulating GSIS. Detailed studies of these proteins and their functions are expected to provide new insights into the molecular mechanisms involved in insulin secretion. [ABSTRACT FROM AUTHOR]

The aim was to investigate the local osteogenic effect of fluvastatin incorporated into a biodegradable gelatin-hydrogel (GH) scaffold. The GH scaffolds were prepared through crosslinking by ultraviolet irradiation followed by freeze-drying. Two circular defects were surgically created on fifteen-week-old male rats calvaria. All defects of each rat were randomly filled with two of three treatments, specifically: fluvastatin incorporated into a GH disk (Flu-GH), distilled water incorporated into a GH disk, and no treatment. New bone formation was quantitatively analyzed after 7, 14, and 28 days using a micro-computed tomography (micro-CT) system, and histologically observed. Evaluation by micro-CT revealed a significant difference in new bone formation among the three kinds of defect. A highly osteogenic effect was observed in the Flu-GH group. The results showed that the fluvastatin incorporated into a biodegradable GH scaffold promoted osteogenesis in rat calvarial bone, indicating its potential for bone regeneration. [ABSTRACT FROM AUTHOR]

We report a simple method, using p53 suppression and nontransforming L-Myc, to generate human induced pluripotent stem cells (iPSCs) with episomal plasmid vectors. We generated human iPSCs from multiple donors, including two putative human leukocyte antigen (HLA)-homozygous donors who match ?20% of the Japanese population at major HLA loci; most iPSCs are integrated transgene-free. This method may provide iPSCs suitable for autologous and allologous stem-cell therapy in the future. [ABSTRACT FROM AUTHOR]

Neurons have highly polarized structures that determine what parts of the soma elaborate the axon and dendrites. However, little is known about the mechanisms that establish neuronal polarity in vivo. Cerebellar Purkinje cells extend a single primary dendrite from the soma that ramifies into a highly branched dendritic arbor. We used the zebrafish cerebellum to investigate the mechanisms by which Purkinje cells acquire these characteristics. To examine dendritic morphogenesis in individual Purkinje cells, we marked the cell membrane using a Purkinje cell-specific promoter to drive membrane-targeted fluorescent proteins. We found that zebrafish Purkinje cells initially extend multiple neurites from the soma and subsequently retract all but one, which becomes the primary dendrite. In addition, the Golgi apparatus specifically locates to the root of the primary dendrite, and its localization is already established in immature Purkinje cells that have multiple neurites. Inhibiting secretory trafficking through the Golgi apparatus reduces dendritic growth, suggesting that the Golgi apparatus is involved in the dendritic morphogenesis. We also demonstrated that in a mutant of an atypical protein kinase C (aPKC), Prkci, Purkinje cells retain multiple primary dendrites and show disrupted localization of the Golgi apparatus. Furthermore, a mosaic inhibition of Prkci in Purkinje cells recapitulates the aPKC mutant phenotype. These results suggest that the aPKC cell autonomously controls the Golgi localization and thereby regulates the specification of the primary dendrite of Purkinje cells. [ABSTRACT FROM AUTHOR]

Direct reprogramming of somatic cells provides an opportunity to generate patient- or disease-specific pluripotent stem cells. Such induced pluripotent stem (iPS) cells were generated from mouse fibroblasts by retroviral transduction of four transcription factors: Oct3/4, Sox2, Klf4 and c-Myc. Mouse iPS cells are indistinguishable from embryonic stem (ES) cells in many respects and produce germline-competent chimeras. Reactivation of the c-Myc retrovirus, however, increases tumorigenicity in the chimeras and progeny mice, hindering clinical applications. Here we describe a modified protocol for the generation of iPS cells that does not require the Myc retrovirus. With this protocol, we obtained significantly fewer non-iPS background cells, and the iPS cells generated were consistently of high quality. Mice derived from Myc− iPS cells did not develop tumors during the study period. The protocol also enabled efficient isolation of iPS cells without drug selection. Furthermore, we generated human iPS cells from adult dermal fibroblasts without MYC. [ABSTRACT FROM AUTHOR]

We evaluated the teratoma-forming propensity of secondary neurospheres (SNS) generated from 36 mouse induced pluripotent stem (iPS) cell lines derived in 11 different ways. Teratoma-formation of SNS from embryonic fibroblast–derived iPS cells was similar to that of SNS from embryonic stem (ES) cells. In contrast, SNS from iPS cells derived from different adult tissues varied substantially in their teratoma-forming propensity, which correlated with the persistence of undifferentiated cells. [ABSTRACT FROM AUTHOR]

Dendrite morphology of neurons provides a structural basis for their physiological characteristics, and is precisely regulated in a cell type-dependent manner. Using a unique transposon-mediated gene transfer system that enables conditional and cell-type specific expression of exogenous genes, we investigated the role of cadherin on dendritic morphogenesis of horizontal cells in the developing chicken retina. We first visualized single horizontal cells by overexpressing membrane-targeted EGFP, and confirmed that there were three subtypes of horizontal cells, the dendritic terminals of which projected to distinct synaptic sites in the outer plexiform layer. Expression of a dominant-negative cadherin decreased the dendritic field size, and perturbed the termination of dendritic processes onto the photoreceptor cells. The cadherin blockade also impaired the accumulation of GluR4, a postsynaptic marker, at the cone pedicles. We thus provide in vivo evidence that cadherin is required for dendrite morphogenesis of horizontal cells and subsequent synapse formation with photoreceptor cells in the vertebrate retina. [ABSTRACT FROM AUTHOR]