Koji Okabayashi

Tissue generation from amphibian animal caps.

Formation of three germ layers is the most important event in early vertebrate development. Animal cap assays can be used to reproduce the in vivo induction of amphibian tissues in order to investigate the differentiation processes that occur in normal embryonic development. Activin treatment strongly and dose-dependently induces various types of mesodermal and endodermal tissue in cultured animal caps. Beating heart, pronephros, pancreas and cartilage can be induced by microsurgical manipulation and simultaneous treatment with activin and other factors. These in vitro induction systems will be helpful for elucidating the mechanisms of tissue induction and organ formation in vertebrate development.

Pdx1-transfected adipose tissue-derived stem cells differentiate into insulin-producing cells in vivo and reduce hyperglycemia in diabetic mice

Insulin-dependent diabetes mellitus (IDDM) is characterized by the rapid development of potentially severe metabolic abnormalities resulting from insulin deficiency. The transplantation of insulin-producing cells is a promising approach for the treatment of IDDM. The transcription factor pancreatic duodenal homeobox 1 (Pdx1) plays an important role in the differentiation of pancreatic beta cells. In this study, the human Pdx1 gene was transduced and expressed in murine adipose tissue-derived stem cells (ASCs). To evaluate pancreatic repair, we used a mouse model of pancreatic damage resulting in hyperglycemia, which involves injection of mice with streptozotocin (STZ). STZ-treated mice transplanted with Pdx1-transduced ASCs (Pdx1-ASCs) showed significantly decreased blood glucose levels and increased survival, when compared with control mice. While stable expression of Pdx1 in ASCs did not induce the pancreatic phenotype in vitro in our experiment, the transplanted stem cells became engrafted in the pancreas, wherein they expressed insulin and C-peptide, which is a marker of insulin-producing cells. These results suggest that Pdx1-ASCs are stably engrafted in the pancreas, acquire a functional beta-cell phenotype, and partially restore pancreatic function in vivo. The ease and safety associated with extirpating high numbers of cells from adipose tissues support the applicability of this system to developing a new cell therapy for IDDM.

Reduction of N-glycolylneuraminic acid in human induced pluripotent stem cells generated or cultured under feeder- and serum-free defined conditions.

Background The successful establishment of human induced pluripotent stem cells (hiPSCs) has increased the possible applications of stem cell research in biology and medicine. In particular, hiPSCs are a promising source of cells for regenerative medicine and pharmacology. However, one of the major obstacles to such uses for hiPSCs is the risk of contamination from undefined pathogens in conventional culture conditions that use serum replacement and mouse embryonic fibroblasts as feeder cells. Methodology/Principal Findings Here we report a simple method for generating or culturing hiPSCs under feeder- and serum-free defined culture conditions that we developed previously for human embryonic stem cells. The defined culture condition comprises a basal medium with a minimal number of defined components including five highly purified proteins and fibronectin as a substrate. First, hiPSCs, which were generated using Yamanakas four factors and conventional undefined culture conditions, adapted to the defined culture conditions. These adapted cells retained the property of self renewal as evaluated morphologically, the expression of self-renewal marker proteins, standard growth rates, and pluripotency as evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo (teratoma formation in immunodeficient mice). Moreover, levels of nonhuman N-glycolylneuraminic acid (Neu5Gc), which is a xenoantigenic indicator of pathogen contamination in human iPS cell cultures, were markedly decreased in hiPSCs cultured under the defined conditions. Second, we successfully generated hiPSCs using adult dermal fibroblast under the defined culture conditions from the reprogramming step. For a long therm culture, the generated cells also had the property of self renewal and pluripotency, they carried a normal karyotype, and they were Neu5Gc negative. Conclusion/Significance This study suggested that generation or adaption culturing under defined culture conditions can eliminate the risk posed by undefined pathogens. This success in generating hiPSCs using adult fibroblast would be beneficial for clinical application.

Ripply2 is essential for precise somite formation during mouse early development

The regions of expression of Ripply1 and Ripply2, presumptive transcriptional corepressors, overlap at the presomitic mesoderm during somitogenesis in mouse and zebrafish. Ripply1 is required for somite segmentation in zebrafish, but the developmental role of Ripply2 remains unclear in both species. Here, we generated Ripply2 knock‐out mice to investigate the role of Ripply2. Defects in segmentation of the axial skeleton were observed in the homozygous mutant mice. Moreover, somite segmentation and expression of Notch2 and Uncx4.1 were disrupted. These findings indicate that Ripply2 is involved in somite segmentation and establishment of rostrocaudal polarity.

In vitro organogenesis from undifferentiated cells in Xenopus

Amphibians have been used for over a century as experimental animals. In the field of developmental biology in particular, much knowledge has been accumulated from studies on amphibians, mainly because they are easy to observe and handle. Xenopus laevis is one of the most intensely investigated amphibians in developmental biology at the molecular level. Thus, Xenopus is highly suitable for studies on the mechanisms of organ differentiation from not only a single fertilized egg, as in normal development, but also from undifferentiated cells, as in the case of in vitro organogenesis. Based on the established in vitro organogenesis methods, we have identified many genes that are indispensable for normal development in various organs. These experimental systems are useful for investigations of embryonic development and for advancing regenerative medicine. Developmental Dynamics 238:1309–1320, 2009.

The Xenopus Bowline/Ripply family proteins negatively regulate the transcriptional activity of T-box transcription factors

Bowline, which is a member of the Xenopus Bowline/Ripply family of proteins, represses the transcription of somitogenesis-related genes before somite segmentation, which makes Bowline indispensable for somitogenesis. Although there are three bowline/Ripply family genes in each vertebrate species, it is not known whether the Bowline/Ripply family proteins share a common role in development. To elucidate their developmental roles, we examined the expression patterns and functions of the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline, and a novel member of this protein family, xRipply3. We found that the expression patterns of bowline and ledgerline overlapped in the presomitic mesoderm (PSM), whereas ledgerline was additionally expressed in the newly formed somites. In addition, we isolated xRipply3, which is expressed in the pharyngeal region. Co-immunoprecipitation assays revealed that Ledgerline and xRipply3 interacted with T-box proteins and the transcriptional co-repressor Groucho/TLE. In luciferase assays, xRipply3 weakly suppressed the transcriptional activity of Tbx1, while Ledgerline strongly suppressed that of Tbx6. In line with the repressive role of Ledgerline, knockdown of Ledgerline resulted in enlargement of expression regions of the somitogenesis-related-genes mespb and Tbx6. Inhibition of histone deacetylase activity increased the expression of mespb, as seen in the Bowline and Ledgerline knockdown experiments. These results suggest that the Groucho-HDAC complex is required for the repressive activity of Bowline/Ripply family proteins during Xenopus somitogenesis. We conclude that although the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline and xRipply3 are expressed differentially, they all act as negative regulators of T-box proteins.

Role of TSC-22 during early embryogenesis in Xenopus laevis.

Transforming growth factor‐β1‐stimulated clone 22 (TSC‐22) encodes a leucine zipper‐containing protein that is highly conserved. During mouse embryogenesis, TSC‐22 is expressed at the site of epithelial–mesenchymal interaction. Here, we isolated Xenopus laevis TSC‐22 (XTSC‐22) and analyzed its function in early development. XTSC‐22 mRNA was first detected in the ectoderm of late blastulae. Translational knockdown using XTSC‐22 antisense morpholino oligonucleotides (XTSC‐22‐MO) caused a severe delay in blastopore closure in gastrulating embryos. This was not due to mesoderm induction or convergent‐extension, as confirmed by whole‐mount in situ hybridization and animal cap assay. Cell lineage tracing revealed that migration of ectoderm cells toward blastopore was disrupted in XTSC‐22‐depleted embryos, and these embryos had a marked increase in the number of dividing cells. In contrast, cell division was suppressed in XTSC‐22 mRNA‐injected embryos. Co‐injection of XTSC‐22‐MO and mRNA encoding p27Xic1, which inhibits cell cycle promotion by binding cyclin/Cdk complexes, reversed aberrant cell division. This was accompanied by rescue of the delay in blastopore closure and cell migration. These results indicate that XTSC‐22 is required for cell movement during gastrulation though cell cycle regulation.

Structural and Functional Changes of Sulfated Glycosaminoglycans in Xenopus laevis during Embryogenesis

Xenopus laevis is an excellent animal for analyzing early vertebrate development. Various effects of glycosaminoglycans (GAGs) on growth factor-related cellular events during embryogenesis have been demonstrated in Xenopus. To elucidate the relationship between alterations in fine structure and changes in the specificity of growth factor binding during Xenopus development, heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS) chains were isolated at four different embryonic stages and their structure and growth factor-binding capacities were compared. The total amounts of both HS and CS/DS chains decreased from the pre-midblastula transition to the gastrula stage, but increased exponentially during the following developmental stages. The length of HS chains was not significantly affected by development, whereas that of CS/DS chains increased with development. The disaccharide composition of GAGs in embryos also changed during development. The degree of sulfation of the HS chains gradually decreased with development. The predominant sulfation position in the CS/DS chains shifted from C4 to C6 of GalNAc during embryogenesis. Growth factor-binding experiments using a BIAcore system demonstrated that GAGs bound growth factors including fibroblast growth factors-1 and -2, midkine, and pleiotrophin, with comparable affinities. These affinities significantly varied during development, although the correlation between the structural alterations of GAGs and the change in the ability to bind growth factors remains to be clarified. The expression of saccharide sequences, which specifically interact with a growth factor, might be regulated during development.

Trb2, a mouse homolog of tribbles, is dispensable for kidney and mouse development

Glomeruli comprise an important filtering apparatus in the kidney and are derived from the metanephric mesenchyme. A nuclear protein, Sall1, is expressed in this mesenchyme, and we previously reported that Trb2, a mouse homolog of Drosophila tribbles, is expressed in the mesenchyme-derived tissues of the kidney by microarray analyses using Sall1-GFP knock-in mice. In the present report, we detected Trb2 expression in a variety of organs during gestation, including the kidneys, mesonephros, testes, heart, eyes, thymus, blood vessels, muscle, bones, tongue, spinal cord, and ganglions. In the developing kidney, Trb2 signals were detected in podocytes and the prospective mesangium of the glomeruli, as well as in ureteric bud tips. However, Trb2 mutant mice did not display any apparent phenotypes and no proteinuria was observed, indicating normal glomerular functions. These results suggest that Trb2 plays minimal roles during kidney and mouse development.

Developmental Potential for Morphogenesis In Vivo and In Vitro

Development is a complex process that involves differentiation into a variety of cell types. In spite of its complexity, the macroscopic pattern and cell types are robust to environmental and developmental perturbations. Even in vitro far from normal developmental conditions, ten normal tissues have been generated from Xenopus animal caps by successive treatment with activin and retinoic acid (RA). To describe both normal development and in vitro organogenesis, we introduce developmental potential following the pioneering study by Waddington. This potential value represents changeability of a cellular state, which decreases toward a local minimum through development. The attraction to a particular cell type through development is described as a process to decrease the potential value to its local minimum. By choosing an explicit potential form as a function of the concentrations of treated activin and RA, the concentration dependence of in vitro organogenesis is reproduced. The potential landscape is shown to have several local minima, each of which represents a stable cell type. This potential also explains why the induction of given tissues requires more treatment of activin at later stages. The consequences of the developmental potential hypothesis encompass the robustness of each tissue generation, the loss of competence through development, and the order of tissues in induction by tissues, which we have confirmed experimentally for in vitro organogenesis. The developmental potential hypothesis for a global description of early development is crucial to understanding the robustness of morphogenesis and explains the achievement of in vitro organogenesis using few molecules as well.