[Show abstract][Hide abstract] ABSTRACT: The periodontal ligament (PDL) contains various cell populations and plays a central role in the maintenance, repair, and regeneration of the periodontium, i.e., tooth-supporting structures. Because primary cells isolated from PDL tissue are heterogeneous, the establishment of an effective isolation method for cells of interest is desired. In the present study, two morphologically distinct cell types were identified in confluent primary cultures derived from rat PDL. To isolate these cell populations, a small piece of filter paper soaked with trypsin-EDTA was placed directly onto the target cell population, enabling the cells to detach from the culture dish. The filter papers were then transferred into fresh culture dishes to establish outgrowth cultures; these two steps constitute the "cell fishing" method. The "fished" cell types were propagated and subcultured for further analyses. In morphological evaluation, immunocytochemical analyses, and reverse transcription-polymerase chain reaction, the isolated cells exhibited a polygonal appearance or a mono- or multinucleated appearance, with a high cytoplasm-to-nucleus ratio, leading to their being characterized as epithelial or myogenic cell populations, respectively. Surprisingly, a notable proportion of the multinuclear cells in the primary and subsequent isolated cultures demonstrated dramatic, spontaneous contractions, a feature typical of skeletal muscle cells. Finally, the isolated cell populations maintained a normal karyotype with a diploid chromosomal number. These results demonstrated that physiological epithelial and skeletal muscle cells can be obtained from primary PDL cultures without artificial induction using growth factors or chemicals, and can be propagated as individual lineage-committed cell populations; the populations consisted of differentiated and progenitor cells that maintained chromosomal stability. This simple, classical culture procedure provides new insights into the biological properties of PDL cells, which are potentially important for the differentiation of tissue or somatic stem cells and for the development of future cell-based therapies for dental and muscular diseases.
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to isolate endothelial cells from tooth buds (unerupted deciduous teeth) of miniature swine. Mandibular molar tooth buds harvested from swine fetuses at fetal days 90-110 were cultured in growth medium supplemented with 15% fetal bovine serum in 100-mm culture dishes until the primary cells outgrown from the tooth buds reached confluence. A morphologically defined set of pavement-shaped primary cells were picked up manually with filter paper containing trypsin/ethylenediamine tetraacetic acid solution and transferred to a separate dish. A characterization of the cellular characteristics and a functional analysis of the cultured cells at passages 3 to 5 were performed using immunofluorescence, a reverse transcriptase polymerase chain reaction assay, a tube formation assay, and transmission electron microscopy. The isolated cells grew in a pavement arrangement and showed the characteristics of contact inhibition upon reaching confluence. The population doubling time was ~48 h at passage 3. As shown by immunocytostaining and western blotting with specific antibodies, the cells produced the endothelial marker proteins such as vascular endothelial cadherin, von Willebrand factor, and vascular endothelial growth factor receptor-2. Observation with time-lapse images showed that small groups of cells aggregated and adhered to each other to form tube-like structures. Moreover, as revealed through transmission electron microscopy, these adherent cells had formed junctional complexes. These endothelial cells from the tooth buds of miniature swine are available as cell lines for studies on tube formation and use in regenerative medical science.
[Show abstract][Hide abstract] ABSTRACT: Background
The aim of this study was to investigate the clinicopathological characteristics of GATA binding protein 3 (GATA3)-positive breast cancers as well as the association of GATA3 expression with response to chemotherapy.Patients and methodsTumor specimens obtained before neoadjuvant chemotherapy [paclitaxel followed by 5-fluorouracil/epirubicin/cyclophosphamide)] from breast cancer patients (n = 130) were subjected to immunohistochemical and mutational analysis of GATA3 and DNA microarray gene expression analysis for intrinsic subtyping.ResultsSeventy-four tumors (57%) were immunohistochemically positive for GATA3. GATA3-positive tumors were significantly more likely to be lobular cancer, estrogen receptor (ER)-positive, progesterone receptor (PgR)-positive, Ki67-negative, and luminal A tumors. Somatic mutations were found in only three tumors. Pathological complete response (pCR) was observed in 8 (11%) GATA3-positive tumors and in 22 (39%) GATA3-negative tumors. Multivariate analysis showed that tumor size, human epidermal growth factor receptor 2 (HER2), and GATA3 were independent predictors of pCR.ConclusionsGATA3-positive breast cancers showed luminal differentiation characterized by high ER expression and were mostly classified as luminal-type tumors following intrinsic subtyping. Interestingly, GATA3 was an independent predictor of response to chemotherapy, suggesting that GATA3 might be clinically useful as a predictor of a poor response to chemotherapy.
[Show abstract][Hide abstract] ABSTRACT: Association of estrogen receptor (ER), progesterone receptor (PR), HER2, Ki67 and 70-gene classifier (70-GC) with a response to paclitaxel (PAC) (n=79) or docetaxel (DOC) (n=55) was investigated in the neoadjuvant setting for breast cancer patients. Sensitivity of breast tumors to PAC, but not to DOC, was found to be significantly associated with ER negativity (P=0.003), PR negativity (P=0.007), and Ki67 positivity (P=0.007). Breast tumors classified into the responders by 70-GC showed a significantly (P=0.005) higher reduction rate to PAC and interestingly a significantly (P=0.009) lower reduction rate to DOC than those classified into the non-responders by 70-GC, suggesting that 70-GC might be useful for the differentiation of PAC-sensitive and DOC-sensitive breast tumors.
Cancer letters 01/2012; 314(2):206-12. · 5.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objectives: The endothelial cell line designated MISEN was established from tooth buds of miniature swine, which is similar to that of human in morphological and physiological characteristics. We try to reveal whether they are capable of forming the tube formation. Methods: The molar tooth buds were obtained from porcine embryo (viviparous age ; 90 days) in aseptic condition. After rinsed several times with Hanks solution, the tooth buds were carried on the bottom of petri dish, and cultured (stationary culture condition)with the growth medium [DMEM/F12 supplemented with 15% FBS, 50 g of penicillin and 50U of streptomycine] in the CO2 incubator. When the outgrowth cells from the tooth buds had become subconfluent, the pavement-shaped cell regions were isolated by colonial cloning method using of the trypsin soaked small filter papers. The isolated cells were cultured on the BD MatrigelTM. Then the cells were observed on the tube formation by video recorder and transmission electron microscope(TEM). The cells also identified with immunohistochemistry. Results: The MISEN cells were grown as a pavement arrangement, and had characteristic of contact inhibition. The karyotype of the cells by G-band staining showed normal diploid. The population doubling time was about 48 hrs. The cells were aggregated each other (after 30 min) and made tube formation (after 60`90min) when they were cultured on the BD Matrigel™. The cells were immunostained with anti-vWF, VE-cadherin and vegfr2. Furthermore, the Weibel-Palade bodies were observed in the cytoplasm by TEM. Conclusion: The MISEN was very important cell line on the studies of the tube formation and regenerative medical science.
[Show abstract][Hide abstract] ABSTRACT: Objectives: Several studies have shown that artificial tooth buds reproduced by natural tooth bud cells can be transplanted into animals to induce tooth formation owing to the expectation of a sufficient blood supply in the animal body. However, this xenotransplantation model carries some risks including infection and immunorejection by the host animals. We developed a novel culture method designated the Organ Engineering Method that involves human periodontal ligament (PDL) cells derived from extracted wisdom teeth, a collagen sponge and newly developed culture media supplemented with embryotrophic factors. Methods: Primary PDL cells outgrowing from PDL tissues were maintained. Confluent PDL cells at passages 2 to 4 were used for organ culture experiments. Tooth crowns (first molars) with no tooth root harvested from newborn mice at postnatal day 5 were cultured using the above culture method for 4 weeks. Results: After 1 week of culture, the root furcation area was formed. After 2 weeks of culture, the two roots had elongated to one-quarter to one-half of the root length from the crown edge. After 4 weeks of culture, newly formed bone was observed below the tooth crown. Soft X-ray and CT analyses revealed the two roots and their surrounding bone and that the tooth roots had a dental pulp space inside of them and a PDL space outside of them. In histological evaluations, three types of hard tissues, namely dentin, cementum and bone, were observed, the collagen fibers were oriented perpendicularly and some epithelial cell rests of Malassez were present in the new PDL. Conclusions: Taken together, the present results indicate that our new culture method enables the engineering of a tooth root/periodontal unit in vitro using practical materials that are clinically available.
[Show abstract][Hide abstract] ABSTRACT: Primary alveolar type of rhabdomyosarcoma (RMS) tumor tissue was collected from the tongue of a 17-year-old Japanese woman and used to successfully establish a rhabdomyosarcoma cell line, which has been designated NUTOS. The chromosomal distribution revealed that the NUTOS cell line was hyper-tetraploid with chromosomal translocation. The cells were grown in Dulbecco's modified eagle medium/F12 supplemented with 15% fetal bovine serum, 0.1% non-essential amino acids solution (NEAA), 50 microg of streptomycin, 50 U/mL of penicillin and 0.25 microg /mL of Fungizone. The NUTOS shapes included small spindles, large spindles and long, thick multinucleated cells. All three cell types were immunostained with anti-desmin antibody, which is a marker protein for middle sized myofilaments. Furthermore, immunocytochemical staining revealed that the cells were positively immunostained with anti-MyoD, myogenin, alpha-sarcomeric actin, myosin and troponin T. Mitotic figures were only observed in the small spindle cells. These cells were coadunated with each other at the lateral portion of the apex of the cells. Subsequently, these cells grew into large multinucleated cells. Autonomic contractions (approximately 20 times/min) were observed in both the large spindle cells and the large multinucleated cells. NUTOS cells incorporated serotonin from the serum in the growth medium. Histopathological observations of the NUTOS cell grafts in the subcutis of nude mice exhibited characteristics similar to those seen for the primary rhabdomyosarcoma of the tongue. Susceptibility tests for the anti-cancer drugs revealed that NUTOS cells were susceptive to cisplatin, paclitaxel, and docetaxel, but not to adriacin.
Human Cell 05/2010; 23(2):65-73. · 1.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abstract Novel cell lines, designated NM78-AM and NM78-MM, have been established from a malignant melanoma of the cheek oral mucosa. NM78-AM cells were spherical, grew in suspension as clusters, and produced no melanin. In contrast, NM78-MM cells were adherent and produced melanin granules. Initially, NM78-AM cells were grown on fibroblast feeder cells or in growth media supplemented with 10% conditioned medium from fibroblasts, but eventually grew in standard growth media alone. NM78-AM cells had interdigitating microvilli and formed cell clusters. They had large nucleoli, desmosomes, lipid droplets, and well-developed Golgi apparatuses. In contrast, NM78-MM cells grew as adherent neuron-like cells. They had large prominent nucleoli, irregular nuclear membranes, a number of mitochondria, well-developed Golgi apparatuses, melanosomes at various stages of development in the cytoplasm, and the cells secreted melanin granules. Projections from these melanotic cells formed anastomoses with each other. NM78-MM cells stained immunofluorescently for internexin, neuron specific enolase, NF-200, and glial fibrillary acidic protein. These cells were severely aneuploid, approximating to triploidy, and had many marker chromosomes. We used a real-time monitoring system to evaluate oxygen concentrations in culture medium to investigate the susceptibility of both cell lines to various anti-cancer drugs. NM78-AM cells were slightly sensitive to actinomycin D, but not to cisplatin, irinotecan, the irinotecan metabolite SN-38, taxol, taxotere, bleomycin and methotrexate; NM78-MM cells were sensitive to cisplatin, and not to taxol, taxotere, carboplatin, and irinotecan. These new cell lines, NM78-AM and NM78-MM, will be very important for the development of new chemotherapeutics for oral malignant melanoma.
Human Cell 02/2010; 23(1):15-25. · 1.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A cell line designated as NEYS was established from ovarian carcinosarcoma (stage IIIc) of a 56-year-old Japanese woman. The extirpated original tumor was carried in growth medium at 0 degrees C to the culture room. The primary culture was done on 20 August 2003. The cell line was composed of angular adhesive cells and showed neoplastic and pleomorphic features, such as bizarre aggregation of chromatin granules, an irregular thickening nuclear membrane and multiple large nucleoli. They grew as multi-layered cultures without contact inhibition. The cells proliferated moderately, and population doubling time was about 56 h. The chromosome number showed an underdiploidy of aneuploidy. The modal chromosome numbers were 37 (36%) and 38 (26%). The cultures produced carcinoembryonic antigen (27.4 ng/mL), carbohydrate antigen 19-9 (210 U/mL), and carbohydrate antigen 125 (526 U/mL). The NEYS cells did not give rise to transplant tumors in nude mice, and showed no susceptibility against cisplatin (CDDP), CPT-11, carboplatin, Paclitaxel, Taxotere and 5-FU. This cell line is useful for studies on the histogenesis of carcinosarcoma and susceptibility of cancer drugs in human ovarian carcinosarcoma. The immunohistochemical and ultrastructual analysis demonstrated that NEYS cells showed epithelial and mesenchymal differentiation, and supported the metaplasis theory as the cause of carcinosarcoma.
Human Cell 09/2009; 22(3):72-80. · 1.41 Impact Factor