To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We demonstrate the allotetraploid origin of X. laevis by partitioning its genome into two homeologous subgenomes, marked by distinct families of “fossil” transposable elements. Based on the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged ~34 million years ago (Mya) and combined to form an allotetraploid ~17–18 Mya. 56% of all genes are retained in two homeologous copies. Protein function, gene expression, and the amount of flanking conserved sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

Induced pluripotent stem cells (iPSCs) can now be produced from various somatic cell (SC) lines by ectopic expression of the four transcription factors. Although the procedure has been demonstrated to induce global change in gene and microRNA expressions and even epigenetic modification, it remains largely unknown how this transcription factor-induced reprogramming affects the total glycan repertoire expressed on the cells. Here we performed a comprehensive glycan analysis using 114 types of human iPSCs generated from five different SCs and compared their glycomes with those of human embryonic stem cells (ESCs; nine cell types) using a high density lectin microarray. In unsupervised cluster analysis of the results obtained by lectin microarray, both undifferentiated iPSCs and ESCs were clustered as one large group. However, they were clearly separated from the group of differentiated SCs, whereas all of the four SCs had apparently distinct glycome profiles from one another, demonstrating that SCs with originally distinct glycan profiles have acquired those similar to ESCs upon induction of pluripotency. Thirty-eight lectins discriminating between SCs and iPSCs/ESCs were statistically selected, and characteristic features of the pluripotent state were then obtained at the level of the cellular glycome. The expression profiles of relevant glycosyltransferase genes agreed well with the results obtained by lectin microarray. Among the 38 lectins, rBC2LCN was found to detect only undifferentiated iPSCs/ESCs and not differentiated SCs. Hence, the high density lectin microarray has proved to be valid for not only comprehensive analysis of glycans but also diagnosis of stem cells under the concept of the cellular glycome.

Background: Vertebrates have numerous lateral asymmetries in the position of their organs, but the molecular basis for the determination of left-right (L-R) asymmetries remains largely unknown. TGFbrelated genes such as lefty and nodal are L-R asymmetrically expressed in developing mouse embryos, and may be involved in L-R determination.

OBJECTIVEDiet composition alters the metabolic states of adipocytes and hepatocytes in diabetes. The effects of dipeptidyl peptidase-4 (DPP-4) inhibition on adipose tissue inflammation and fatty liver have been obscure. We investigated the extrapancreatic effects of DPP-4 inhibition on visceral fat and the liver.RESEARCH DESIGN AND METHODSWe investigated diet-induced metabolic changes in β-cell–specific glucokinase haploinsufficient (Gck+/−) diabetic mice. We challenged animals with a diet containing a combination of sucrose and oleic acid (SO) or sucrose and linoleic acid (SL). Next, we assessed the effects of a DPP-4 inhibitor, des-fluoro-sitagliptin, on adipose tissue inflammation and hepatic steatosis.RESULTSThe epididymal fat weight and serum leptin level were significantly higher in Gck+/− mice fed SL than in mice fed SO, although no significant differences in body weight or adipocyte size were noted. Compared with SO, SL increased the numbers of CD11c+ M1 macrophages and CD8+ T-cells in visceral adipose tissue and the expression of E-selectin, P-selectin, and plasminogen activator inhibitor-1 (PAI-1). DPP-4 inhibition significantly prevented adipose tissue infiltration by CD8+ T-cells and M1 macrophages and decreased the expression of PAI-1. The production of cytokines by activated T-cells was not affected by DPP-4 inhibition. Furthermore, DPP-4 inhibition prevented fatty liver in both wild-type and Gck+/− mice. DPP-4 inhibition also decreased the expressions of sterol regulatory element–binding protein-1c, stearoyl-CoA desaturase-1, and fatty acid synthase, and increased the expression of peroxisome proliferator–activated receptor-α in the liver.CONCLUSIONSOur findings indicated that DPP-4 inhibition has extrapancreatic protective effects against diet-induced adipose tissue inflammation and hepatic steatosis.

Insulin-like growth-factor-binding proteins (IGFBPs) bind to and modulate the actions of insulin-like growth factors (IGFs) 1 . Although some of the actions of IGFBPs have been reported to be independent of IGFs, the precise mechanisms of IGF-independent actions of IGFBPs are largely unknown 1,2 . Here we report a previously unknown function for IGFBP-4 as a cardiogenic growth factor. IGFBP-4 enhanced cardiomyocyte differentiation in vitro, and knockdown of Igfbp4 attenuated cardiomyogenesis both in vitro and in vivo. The cardiogenic effect of IGFBP-4 was independent of its IGF-binding activity but was mediated by the inhibitory effect on canonical Wnt signalling. IGFBP-4 physically interacted with a Wnt receptor, Frizzled 8 (Frz8), and a Wnt co-receptor, lowdensity lipoprotein receptor-related protein 6 (LRP6), and inhibited the binding of Wnt3A to Frz8 and LRP6. Although IGF-independent, the cardiogenic effect of IGFBP-4 was attenuated by IGFs through IGFBP-4 sequestration. IGFBP-4 is therefore an inhibitor of the canonical Wnt signalling required for cardiogenesis and provides a molecular link between IGF signalling and Wnt signalling.The heart is the first organ to form during embryogenesis, and abnormalities in this process result in congenital heart diseases, the most common cause of birth defects in humans 3 . Molecules that mediate cardiogenesis are of particular interest because of their potential use for cardiac regeneration 4,5 . Previous studies have shown that soluble growth factors such as bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), Wnts and Wnt inhibitors mediate the tissue interactions that are crucial for cardiomyocyte specification 3,4 . We proposed that there might be additional soluble factors that modulate cardiac development and/or cardiomyocyte differentiation.P19CL6 cells differentiate into cardiomyocytes with high efficiency in the presence of 1% dimethylsulphoxide (DMSO) 6 . We cultured P19CL6 cells with culture media conditioned by various cell types in the absence of DMSO, and screened the cardiogenic activity of the conditioned media. The extent of cardiomyocyte differentiation was assessed by the immunostaining with MF20 monoclonal antibody that recognizes sarcomeric myosin heavy chain (MHC). Among the several cell types tested, culture media conditioned by a murine stromal cell line OP9 induced cardiomyocyte differentiation of P19CL6 cells without DMSO treatment (Fig. 1a, left and middle panels). Increased MF20-positive area was accompanied by the induction of cardiac marker genes such as aMHC, Nkx2.5 and GATA-4, and by the increased protein levels of cardiac troponin T (cTnT) (Fig. 1a, right panel). In contrast, culture media conditioned by COS7 cells, mouse embryonic fibroblasts, NIH3T3 cells, HeLa cells, END2 cells (visceral endoderm-like cells), neonatal rat cardiomyocytes and neonatal rat cardiac fibroblasts did not induce cardiomyocyte differentiation of P19CL6 cells in the absence of DMSO (Fig. 1a and data not shown). From these observations, we p...

In the present study, we demonstrated that insulin is produced not only in the mammalian pancreas but also in adult neuronal cells derived from the hippocampus and olfactory bulb (OB). Paracrine Wnt3 plays an essential role in promoting the active expression of insulin in both hippocampal and OB-derived neural stem cells. Our analysis indicated that the balance between Wnt3, which triggers the expression of insulin via NeuroD1, and IGFBP-4, which inhibits the original Wnt3 action, is regulated depending on diabetic (DB) status. We also show that adult neural progenitors derived from DB animals retain the ability to give rise to insulin-producing cells and that grafting neuronal progenitors into the pancreas of DB animals reduces glucose levels. This study provides an example of a simple and direct use of adult stem cells from one organ to another, without introducing additional inductive genes.

We recently reported that induced pluripotent stem cells (iPSCs) prepared from different human origins acquired similar glycan profiles to one another as well as to human embryonic stem cells. Although the results strongly suggested attainment of specific glycan expressions associated with the acquisition of pluripotency, the detailed glycan structures remained to be elucidated. Here, we perform a quantitative glycome analysis targeting both N-and Olinked glycans derived from 201B7 human iPSCs and human dermal fibroblasts as undifferentiated and differentiated cells, respectively. Overall, the fractions of high mannose-type N-linked glycans were significantly increased upon induction of pluripotency. Moreover, it became evident that the type of linkage of Sia on N-linked glycans was dramatically changed from ␣-2-3 to ␣-2-6, and the expression of ␣-1-2 fucose and type 1 LacNAc structures became clearly apparent, while no such glycan epitopes were detected in fibroblasts. The expression profiles of relevant glycosyltransferase genes were fully consistent with these results. These observations indicate unambiguously the manifestation of a "glycome shift" upon conversion to iPSCs, which may not merely be the result of the initialization of gene expression, but could be involved in a more aggressive manner either in the acquisition or maintenance of the undifferentiated state of iPSCs. Molecular

SummaryThe application of stem-cell-based therapies in regenerative medicine is hindered by the tumorigenic potential of residual human pluripotent stem cells. Previously, we identified a human pluripotent stem-cell-specific lectin probe, called rBC2LCN, by comprehensive glycome analysis using high-density lectin microarrays. Here we developed a recombinant lectin-toxin fusion protein of rBC2LCN with a catalytic domain of Pseudomonas aeruginosa exotoxin A, termed rBC2LCN-PE23, which could be expressed as a soluble form from the cytoplasm of Escherichia coli and purified to homogeneity by one-step affinity chromatography. rBC2LCN-PE23 bound to human pluripotent stem cells, followed by its internalization, allowing intracellular delivery of a cargo of cytotoxic protein. The addition of rBC2LCN-PE23 to the culture medium was sufficient to completely eliminate human pluripotent stem cells. Thus, rBC2LCN-PE23 has the potential to contribute to the safety of stem-cell-based therapies.