Takashi Ariizumi

A model system for organ engineering: transplantation of in vitro induced embryonic kidney.

Naturwissenschaften 86 (1999) Q Springer-Verlag 1999 criminate colours independently of the intensity and that they use the red receptor when they do so. It thus seems likely that Papilio colour vision is at least tetrachromatic, including the three receptor types shared by most insects as well as the red receptor. To test how many dimensions the butterfly colour space really has, more experiments are needed. It might, however, be very difficult to test for the influences of the three receptors sensitive in the short wavelength region since their sensitivity maxima lie very close together.

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.

In vitro control of organogenesis and body patterning by activin during early amphibian development

In the process of amphibian development, an embryonic body plan is established through cell division, sequential gene expression, morphogenesis and cell differentiation. The mechanism of body patterning is complex and includes multiple induction events. Activin, a TGF-beta family protein, can induce several kinds of mesodermal and endodermal tissues in animal cap explants in a dose-dependent manner. In a recent study of the role of activin in organogenesis, we succeeded in raising a beating heart by treating animal caps with a high concentration of activin. Activin also participates in kidney organogenesis in combination with retinoic acid. An embryonic kidney induced by activin and retinoic acid in vitro can function in vivo when it is transplanted into a larva in which pronephros rudiments have already been removed. Further, the activin-treated animal caps clearly show organizer actions that are closely related to body patterning along the anteroposterior axis. These experiments will help to serve as a model system for understanding organogenesis and body patterning at the cellular and molecular levels.

Head and trunk-tail organizing effects of the gastrula ectoderm of Cynops pyrrhogaster after treatment with activin A

Differentiation tendency and the inducing ability of the presumptive ectoderm of newt early gastrulae were examined after treatment with activin A at a high concentration (100 ng/ml). The activin-treated ectoderm differentiated preferentially into yolk-rich endodermal cells. Combination explants consisting of three pieces of activin-treated ectoderm formed neural tissues and axial mesoderm along with endodermal cells. However, the neural tissue was poorly organized and never showed any central nervous system characteristics. When the activin-treated ectoderm was sandwiched between two sheets of untreated ectoderm, the sandwich explants differentiated into trunk-tail or head structures depending on the duration of preculture of activin-treated ectoderm in Holtfreters solution. Short-term (0–5 h) precultured ectoderm induced trunk-tail structures accompanied by axial organs, alimentary canal and beating heart. The arrangement of the explant tissues and organs was similar to that of normal embryos. However, archencephalic structures, such as forebrain and eye, were lacking or deficient. On the other hand, long-term (10–25 h) precultured ectoderm induced archencephalic structures in addition to axial organs. Lineage analysis of the sandwich explants using fluorescent dyes revealed that the activin-treated ectoderm mainly differentiated into endodermal cells and induced axial mesoderm and central nervous system in the untreated ectoderm. These results suggest that activin A is one of the substances involved in triggering endodermal differentiation and that the presumptive ectoderm induced to form endoderm displays “trunk-tail organizer” or “head organizer” effects, depending on the duration of preculture.

In Vitro Control of the Embryonic Form of Xenopus laevis by Activin A: Time and Dose-Dependent Inducing Properties of Activin-Treated Ectoderm

The inducing properties of activin‐treated ectoderm of Xenopus laevis were examined by the preculture and sandwich culture methods. Presumptive ectodermal sheets of the late blastula were treated with 10–100 ng/ml of activin A and precultured for 0–7 hr in Steinbergs solution. They were then sandwiched between two sheets of ectoderm from other late blastulae. Ectoderm precultured for a short term induced trunk‐tail structures, whereas that precultured for a long term induced head structures in addition to trunk‐tail structures. These time‐dependent changes in inducing properties occurred more rapidly when the concentration of activin A was higher. These results suggest that the activin‐treated ectoderm functioned as a “head organizer” or “trunk‐tail organizer” depending upon the concentration of activin A and the duration of preculture.

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.

Activin A induces craniofacial cartilage from undifferentiated Xenopus ectoderm in vitro

Activin A has potent mesoderm-inducing activity in amphibian embryos and induces various mesodermal tissues in vitro from the isolated presumptive ectoderm. By using a sandwich culture method established to examine activin A activity, we previously demonstrated that activin-treated ectoderm can function as both a head and trunk-tail organizer, depending on the concentration of activin A. By using activin A and undifferentiated presumptive ectoderm, it is theoretically possible to reproduce embryonic induction. Here, we test this hypothesis by studying the induction of cartilage tissue by using the sandwich-culture method. In the sandwiched explants, the mesenchymal cell condensation expressed type II collagen and cartilage homeoprotein-1 mRNA, and subsequently, cartilage was induced as they are in vivo. goosecoid (gsc) mRNA was prominently expressed in the cartilage in the explants. Xenopus distal-less 4 (X-dll4) mRNA was expressed throughout the explants. In Xenopus embryos, gsc expression is restricted to the cartilage of the lower jaw, and X-dll4 is widely expressed in the ventral head region, including craniofacial cartilage. These finding suggest that the craniofacial cartilage, especially lower jaw cartilage, was induced in the activin–treated sandwiched explants. In addition, a normal developmental pattern was recapitulated at the histological and genetic level. This work also suggests that the craniofacial cartilage-induction pathway is downstream of activin A. This study presents a model system suitable for the in vitro analysis of craniofacial cartilage induction in vertebrates.

Control of the Embryonic Body Plan by Activin during Amphibian Development

Abstract Embryonic induction plays an important role in establishing the fundamental body plan during early amphibian development. The factors mediating this embryonic induction have, however, only recently been discovered. In the mid-1980s, certain peptide growth factors belonging to the FGF and TGF-β families were found to have a mesoderm-inducing effect on isolated Xenopus blastula ectoderm. The study of embryonic induction subsequently expanded rapidly and knowledge at the molecular level has gradually accumulated. One of these peptide growth factors, activin, a member of the TGF-β superfamily, is present maternally in the Xenopus early embryo and induces various mesodermal and endodermal tissues in isolated presumptive ectoderm. After exposure of presumptive ectoderm to activin, many genes are expressed in the same manner as in normal embryogenesis. Ectoderm treated with activin can induce a complete secondary embryo, the same as the organizer does in transplantation experiments. These findings suggest that activin is one of the first induction signals responsible for establishing the embryonic body plan in early amphibian development. In this article we shall review to what extent we can control the embryonic body plan in vitro, referring to some significant findings in this field.

Cytochalasin B inhibits morphogenetic movement and muscle differentiation of activin‐treated ectoderm in Xenopus

Xenopus ectodermal explants (animal caps) begin to elongate after treatment with the mesoderm inducing factor activin A. This phenomenon mimics the convergent extension of dorsal mesoderm during gastrulation. To analyze the relationship between elongation movement and muscle differentiation, animal caps were treated with colchicine, taxol, cytochalasin B and hydroxyurea (HUA)/aphidicolin following activin treatment. Cytochalasin B disrupted the organization of actin filaments and inhibited the elongation of the activin‐treated explants. Muscle differentiation was also inhibited in these explants at the histologic and molecular levels. Colchicine and taxol, which are known to affect microtubule organization, had little effect on elongation of the activin‐treated explants. Co‐treatment with HUA and aphidicolin caused serious damage on the explants and they did not undergo elongation. These results suggest that actin filaments play an important role in the elongation movement that leads to muscle differentiation of activin‐treated explants.

Isolation and Differentiation of Xenopus Animal Cap Cells

Xenopus is used as a model animal for investigating the inductive events and organogenesis that occur during early vertebrate development. Given that they are easy to obtain in high numbers and are relatively large in size, Xenopus embryos are excellent specimens for performing manipulations such as microinjection and microsurgery. The animal cap, which is the area around the animal pole of the blastula, is destined to form the ectoderm during normal development. However, these cells retain pluripotentiality and upon exposure to specific inducers, the animal cap can differentiate into neural, mesodermal, and endodermal tissues. In this sense, the cells of the animal cap are equivalent to mammalian embryonic stem cells. In this unit, the isolation and differentiation of animal cap cells, the so-called animal cap assay, is described. Useful methods for analyzing the mechanism of animal cap differentiation at the molecular level are also described.