Integrins regulate mouse embryonic stem cell self-renewal.

Extracellular matrix (ECM) components regulate stem-cell behavior, although the exact effects elicited in embryonic stem (ES) cells are poorly understood. We previously developed a simple, defined, serum-free culture medium that contains leukemia inhibitory factor (LIF) for propagating pluripotent mouse embryonic stem (mES) cells in the absence of feeder cells. In this study, we determined the effects of ECM components as culture substrata on mES cell self-renewal in this culture medium, comparing conventional culture conditions that contain serum and LIF with gelatin as a culture substratum. mES cells remained undifferentiated when cultured on type I and type IV collagen or poly-D-lysine. However, they differentiated when cultured on laminin or fibronectin as indicated by altered morphologies, the activity of alkaline phosphatase decreased, Fgf5 expression increased, and Nanog and stage-specific embryonic antigen 1 expression decreased. Under these conditions, the activity of signal transducer and activator of transcription (STAT)3 and Akt/protein kinase B (PKB), which maintain cell self-renewal, decreased. In contrast, the extracellular signal-regulated kinase (ERK)1/2 activity, which negatively controls cell self-renewal, increased. In the defined conditions, mES cells did not express collagen-binding integrin subunits, but they expressed laminin- and fibronectin-binding integrin subunits. The expression of some collagen-binding integrin subunits was downregulated in an LIF concentration-dependent manner. Blocking the interactions between ECM and integrins inhibited this differentiation. Conversely, the stimulation of ECM-integrin interactions by overexpressing collagen-binding integrin subunits induced differentiation of mES cells cultured on type I collagen. The results of the study indicated that inactivation of the integrin signaling is crucial in promoting mouse embryonic stem cell self-renewal. Disclosure of potential conflicts of interest is found at the end of this article.

Modulation of activin A-induced differentiation in vitro by vascular endothelial growth factor in Xenopus presumptive ectodermal cells.

We have previously demonstrated that activin A at low concentrations induced ventral mesoderm including blood-like cells from Xenopus animal caps and that beating heart could be also induced from animal caps treated with 100 ng/ml activin A, suggesting that activin A might be involved in cardiac vasculogenesis. A vascular endothelial growth factor (VEGF) is a powerful mitogen for endothelial cells and is an inducer and regulator of angiogenesis. However, VEGF function in Xenopus development is not clearly identified. In this study, we determined the effect of VEGF on activin A-induced differentiation of animal cap. The VEGF induced duct-like structure composed of Flk-1-positive cells together with the induction of nonvascular tissues, such as neural tissues. This histological result was coincident with our reverse transcriptase-polymerase chain reaction analysis that VEGF together with activin A promoted the expression of Xenopus N-CAM and Xenopus brachyury. This study suggests that VEGF has additional biological activities besides angiogenesis, and arises a different function that VEGF induces stroma cell migration or recruitment that are required for blood vessel formation. This differentiation system will aid in the understanding of angiogenesis during early development.

Leukemia inhibitory factor as an anti-apoptotic mitogen for pluripotent mouse embryonic stem cells in a serum-free medium without feeder cells.

We have developed a serum-free medium, designated ESF7, in which leukemia inhibitory factor (LIF) clearly stimulated murine embryonic stem (ES) cell proliferation accompanied by increased expression of nanog and Rex-1 and decreased FGF-5 expression. These effects were dependent on the concentration of LIF. The ES cells maintained in ESF7 medium for more than 2 yr retained an undifferentiated phenotype, as manifested by the expression of the transcription factor Oct-3/4, the stem cell marker SSEA-1, and alkaline phosphatase. Withdrawal of LIF from ESF7 medium resulted in ES cell apoptosis. Addition of serum to ESF7 medium promoted ES cell differentiation. Addition of BMP4 promoted ES cell differentiation into simple epithelial-like cells. In contrast, FGF-2 promoted ES cell differentiation into neuronal and glial-like cells. Under serum-free culture conditions, LIF was sufficient to stimulate cell proliferation, it inhibited cell differentiation, and it maintained self-renewal of ES cells. Because this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent ES cells in vitro, it will allow the elucidation of ES cell responses to growth factors under defined conditions.

Induction of tooth and eye by transplantation of activin A-treated, undifferentiated presumptive ectodermal Xenopus cells into the abdomen.

Activin A can induce the Xenopus presumptive ectoderm (animal cap) to form different types of mesoderm and endoderm at different concentrations and the animal cap treated with activin can function as an organizer during early development. The dissociated Xenopus animal cap cells treated with activin form an aggregate and it develops into various tissues in vitro. In this study, to induce jaw cartilage from undifferentiated cells effectively, we developed a culture method to manipulate body patterning in vitro, using activin A and dissociated animal cap cells. An aggregate consisting only of activin A-treated dissociated cells developed into endodermal tissues. However, when activin A-treated cells were mixed with untreated cells at a ratio of 1:5, the aggregate developed cartilage with the maxillofacial regional marker genes, goosecoid, Xenopus Distal-less 4 and X-Hoxa2. When this aggregate was transplanted into the abdominal region of host embryos, maxillofacial structures containing cartilage and eye developed. We raised these embryos to adulthood and found that tooth germ had developed in the transplanted tissue. Here, we show the induction of jaw cartilage, tooth germ and eye structures from animal caps using activin A in the aggregation culture method. This differentiation system will help to promote a better understanding of the regulating mechanisms of body patterning and tooth induction in vertebrates.

Activin-like signaling activates Notch signaling during mesodermal induction.

Both activin-like signaling and Notch signaling play fundamental roles during early development. Activin-like signaling is involved in mesodermal induction and can induce a broad range of mesodermal genes and tissues from prospective ectodermal cells (animal caps). On the other hand, Notch signaling plays important roles when multipotent precursor cells achieve a specific cell fate. However, the relationship between these two signal pathways is not well understood. Here, we show that activin A induces Delta-1, Delta-2 and Notch expression and then activates Notch signaling in animal caps. Also, in vivo, ectopic activin-like signaling induced the ectopic expression of Delta-1 and Delta-2, whereas inhibition of activin-like signaling abolished the expression of Delta-1 and Delta-2. Furthermore, we show that MyoD, which is myogenic gene induced by activin A, can induce Delta-1 expression. However, MyoD had no effect on Notch expression, and inhibited Delta-2 expression. These results indicated that activin A induces Delta-1, Delta-2 and Notch by different cascades. We conclude that Notch signaling is activated when activin-like signaling induces various tissues from homogenous undifferentiated cells.

Long-term culture of Xenopus presumptive ectoderm in a nutrient-supplemented culture medium.

Animal cap assay is a useful experimental model for investigating the activity of inducers in amphibian development. This assay has revealed that activin A is a potent mesoderm-inducing factor. However, it has been very difficult to induce highly differentiated tissues such as cartilage in a 3-4 day culture period. It was recently reported that jaw cartilage was induced in vitro in an animal cap that had been cultured for 14 days in Steinberg's solution using the sandwich culture method and activin A. Under these conditions, necrosis was occasionally observed in the explants. In this study, we have achieved long-term animal cap cultures in a nutrient-supplemented culture medium designated RDX. This medium was made by modifying the saline concentration of the RD medium previously developed as a basal medium for the serum-free culture of various kinds of mammalian cells. The explants cultured in RDX grew more vigorously compared with those in Steinberg's solution. RDX medium promoted a wider variety of tissue induction and gene expression in the animal caps than Steinberg's solution, and also increased the frequency of cartilage induction. Therefore, the supplemental nutrients may support and promote the differentiation of cartilage. This long-term culture method using RDX medium is useful for studying the differentiation of tissues or organs such as cartilage in vitro.

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 were 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.