Miho Furue

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.

LEUKEMIA INHIBITORY FACTOR AS AN ANTI-APOPTOTIC MITOGEN FOR PLURIPOTENT MOUSE EMBRYONIC STEM CELLS IN A SERUM-FREE MEDIUM WITHOUT FEEDER CELLS

SummaryWe 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 MBP4 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.

Effects of hepatocyte growth factor (HGF) and activin a on the morphogenesis of rat submandibular gland-derived epithelial cells in serum-free collagen gel culture

SummaryTo study the mechanisms of morphogenesis in salivary gland regeneration, we have established the RSMG-1 cell line derived from submandibular gland (SMG) of 10-wk-old Wistar female rats in serum-free culture. Our finding that RSMG-1 cells originated from duct cells was based on morphology and immunohistochemical results. In three-dimensional serum-free collagen gel culture, HGF induced branching morphogenesis of RSMG-1 cells. Histological examination revealed that HGF-induced branching structure exhibited well-formed lumina. This morphology closely resembles that found in vivo. The cells also expressed activin A. Exogenously added activin A at a high concentration reduced HGF-induced branching morphogenesis. These findings suggest that the morphogenesis of the salivary gland is modulated by HGF and activin A. Our results show that the RSMG-1 cell line may be useful in studies of salivary gland regeneration.

Primitive neuroectodermal tumor cell lines derived from a metastatic pediatric tumor

Dear Editor: We have established two cell lines in serum-free culture from different stages of a metastatic primitive neuroectodermal tumor (PNET) isolated from a 2-year-old patient. Among human brain tumors, PNETs are rare malignant multipotent pediatric neoplasia capable of exhibiting all stages of neurogenic maturation (5,15). This tumor, diagnosed as progressive PNET, was first found in a retroperitoneal mass; it recurred in the perirectal space and metastasized as a perianal lesion. Two specimens of the tumor were retrieved from the perirectal space and the perianal lesion. A cell line derived from a perirectal lesion was designated RS-1, and the second cell line derived from a perianal mass was designated RS-2. Tumor specimens were cut, minced, and seeded in 60 mm plastic dishes (Falcon, Oxnard, CA). The cells were maintained in a humidified atmosphere of 5% CO2 at 37 ° C in RD medium supplemented with 6 factors (6F) (17). 6 factors consisted of 10 #g/ml bovine insulin, 5 #g/ml human transferrin, 50/. tg/ml fatty acid-free bovine serum albumin, 10 #M 2-mercaptoethanol, 10 #M 2-aminoethanol, and 10 nM sodium selenite (all from Sigma, St. Louis, Mo.). Each factor was made as a sterile 100)< concentrate and stored at 4 ° C. RD medium was a 1:1 mixture (by volume) of RPMI 1640 medium (Kyokuto, Tokyo, Japan) and Dulbeccos modified Eagles medium (Kyokuto), to which was added 0.01% sodium pyruvate (Sigma), 2.2 g/liter sodium bicarbonate (Wako Chemicals Tokyo, Japan), 15 mM HEPES (Dojin, Tokyo, Japan), streptomycin and kanamycin solution (GIBCO Laboratories, Grand Island, NY). There are reports in the literature of PNET cells being cultured for short periods of time to determine chromosome aberrations or to examine nerve growth factor (NGF) receptor expression (1,6,7,10, 11,19,22,24,25), but long-term culture of such cells has not been described. Among several culture conditions tested, RD medium supplemented with 6 factors gave the greatest degree of cell proliferation. The doubling time of RS-1 was 48 h, and the doubling time of RS-2 was 24 h (Fig. 1). Ewings sarcoma is closely related to PNET histogenically (8,12-14,20), and it is sometimes difficult to distinguish PNET from Ewings sarcoma. Under the light microscope RS-1 and RS-2 cells appeared small in size with short neurites. Immunohistocbemical examination revealed that RS-1 cells expressed neuron-specific enolase protein (NSE) (Fig. 2 A), neurofilament (NF) (Fig. 2 B), S-100 protein (S-100) (Fig. 2 C), and vimentin (Fig. 2 D). Expression of glial fibillary acidic protein (GFAP) was not found. According to an old classification system, NSE expression is significant in making a diagnosis of PNET. According to a newly proposed classification system, expression of two different neural markers can lead to a diagnosis of PNET (5,15). Our results showed that RS-1 cells had a typical PNET phenotype. Although RS-2 cells expressed vimentin (Fig. 2 H) and S-100 (Fig. 2 G), which is a neural marker,

Induction of Cells Expressing Vascular Endothelium Markers from Undifferentiated Xenopus Presumptive Ectoderm by Co-treatment with Activin and Angiopoietin-2

Abstract Activin is a potent inducer of mesoderm in amphibian embryos. We previously reported that low concentrations of activin could induce the formation of blood cells from Xenopus explants (animal caps). Both hematopoietic and vascular endothelial cell lineages are believed to share a common precursor, termed hemangioblasts. In this study, we tried to induce differentiation of vascular endothelial cells in aggregates derived from Xenopus animal caps. Aggregates formed from cells that were co-treated with activin and angiopoietin-2 expressed the vascular endothelial markers, X-msr, Xtie2 and Xegfl7. However, none of these aggregates expressed the hematopoietic marker genes, globin alpha T3, alpha T5, alpha A or GATA-1. We used microarray analysis to compare the gene expression profiles of aggregates treated with activin alone or with activin and angiopoietin. The combination, but not activin alone, induced expression of vascular-related genes such as Xl-fli and VEGF. These results demonstrate that treatment of dissociated animal cap cells with activin and angiopoietin-2 can induce differentiation of endothelial cells, and provides a promising model system for the in vitro study of blood vessel induction in vertebrates.

Atrophic Dermatofibrosarcoma protuberans: A Case Report and Review of the Literature

Dermatofibrosarcoma protuberans is not a difficult tumor to recognize because of its characteristic clinical appearance, although some unusual variants have been reported. We describe the atrophic variant of dermatofibrosarcoma protuberans in a 21-year-old female. The lesion was a smooth-surfaced, oval depression on the left subclavicular area, with a violaceous plaque at the center. The suspected clinical diagnosis did not include fibrous tumors, although histological examination showed the typical picture of dermatofibrosarcoma protuberans. Positive CD34 staining was also helpful in the diagnosis. We review 14 cases of the atrophic variant of dermatofibrosarcoma protuberans in the literature. Dermatologists should be aware of this uncommon but characteristic appearance of atrophic dermatofibrosarcoma protuberans.

Serum-free culture conditions for serial subculture of undifferentiated PC12 cells

PC12 cells, a widely used model neuronal cell line, are usually cultured in serum-supplemented medium. This report describes a serum-free medium for the culture of PC12 cells. PC12 cells grown in the two media types had similar growth rates and released dopamine in response to high potassium-induced calcium elevation. However, the levels of dopamine and of dopamine release in cells cultured in the serum-free medium were less than 10% of that in cells cultured in serum-supplemented medium. Dopamine levels recovered within 10 days if cells were returned to serum-supplemented medium, but dopamine release could not be recovered. Nerve growth factor (NGF) induced similar responses in PC12 cells cultured in both media, including phosphorylation of extracellular signal-regulated protein kinases and neurite extension. Transferrin was necessary for survival of neurite-bearing PC12 cells subcultured in serum-free medium and insulin promoted the cells proliferation. Ten days culture with NGF produced a similar increase in neurofilament expression and acetylcholinesterase activity in both media. These results suggest that PC12 in the hormonally defined serum-free media are qualitatively the same as those cultured in serum-supplemented media, and therefore this new culture protocol should enable more precise studies of PC12 cells culture in the absence of confounding unknown factors.

Experimental Split Cord Malformations

Objective: To induce experimental split cord malformations (SCMs) produced through the surgical induction of a dorsal midline fistula. Methods: In addition, the theory of embryogenesis of SCMs was verified by examining the developmental process of this experimentally induced anomaly. In Cynopus pyrrhogaster (amphibian) embryos (stage 18), the neural plate and notochord were split regionally to construct a fistula that appeared to be the ectopic neurenteric canal. Following this procedure, the embryonic development was traced morphologically and histologically. Results: Following the incubation and breeding period, split cord malformation was observed in some animals. Scoliosis, spina bifida, vertebral anomaly and subcutaneous manifestations were also observed with SCMs. Conclusions: The observations made in these experimentally induced SCMs are consistent with the findings in human SCMs. We report an experimental animal model of split cord malformation, in which double spinal cords were developed in the spinal canal. In addition, we examined the embryogenesis of SCMs. This study indicates that SCMs may arise through a process of dorsal midline fistula of the neural plate.

Notch signaling modulates the nuclear localization of carboxy-terminal-phosphorylated smad2 and controls the competence of ectodermal cells for activin A.

Loss of mesodermal competence (LMC) during Xenopus development is a well known but little understood phenomenon that prospective ectodermal cells (animal caps) lose their competence for inductive signals, such as activin A, to induce mesodermal genes and tissues after the start of gastrulation. Notch signaling can delay the onset of LMC for activin A in animal caps [Coffman, C.R., Skoglund, P., Harris, W.A., Kintner, C.R., 1993. Expression of an extracellular deletion of Xotch diverts cell fate in Xenopus embryos. Cell 73, 659-671], although the mechanism by which this modulation occurs remains unknown. Here, we show that Notch signaling also delays the onset of LMC in whole embryos, as it did in animal caps. To better understand this effect and the mechanism of LMC itself, we investigated at which step of activin signal transduction pathway the Notch signaling act to affect the timing of the LMC. In our system, ALK4 (activin type I receptor) maintained the ability to phosphorylate the C-terminal region of smad2 upon activin A stimulus after the onset of LMC in both control- and Notch-activated animal caps. However, C-terminal-phosphorylated smad2 could bind to smad4 and accumulate in the nucleus only in Notch-activated animal caps. We conclude that LMC was induced because C-terminal-phosphorylated smad2 lost its ability to bind to smad4, and consequently could not accumulate in the nucleus. Notch signal activation restored the ability of C-terminal-phosphorylated smad2 to bind to smad4, resulting in a delay in the onset of LMC.

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 Steinbergs 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 Steinbergs solution. RDX medium promoted a wider variety of tissue induction and gene expression in the animal caps than Steinbergs 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.