Types of cell culture inserts affect cell crosstalk between co-cultured macrophages and adipocytes.

Cell culture inserts offer an in vivo-like microenvironment to investigate cell-cell interactions between co-cultivated cells. However, it is unclear if types of inserts affect cell crosstalk. Here, we developed an environment-friendly cell culture insert, XL-insert, which can reduce plastic waste with lower cost. We compared XL insert with two types of commercial disposable culture inserts, Koken® insert with atelocollagen membrane (Col-inserts) and Falcon® inserts with plastic membrane (PET-inserts) on cell-cell interactions in co-cultivated THP-1 macrophages and OP9 adipocytes. Scanning electron microscope, immunoassay and imaging analysis showed that among three types of inserts, XL-inserts allowed cytokines from co-cultivated macrophages and adipocytes to diffuse freely and offered preferable in vivo-like microenvironment for cell-cell interactions. PET-inserts showed limitations for intercellular communication due to some pores being blocked by somas on the membrane that caused much lower permeability for cytokines passing through. Col-inserts blocked large sized cytokines but allowed small sized molecules to permeate resulting in improved lipid accumulation and adiponectin secretion in OP9 adipocytes. Taken together, our data demonstrated that membrane type and pore size on the membrane affect the cross-talk between co-cultivated cells very differently. Some previous co-culture studies might have different results if the inserts were changed.

Enzyme-Digested Edible Bird's Nest (EBND) Prevents UV and Arid Environment-Induced Cellular Oxidative Stress, Cell Death and DNA Damage in Human Skin Keratinocytes and Three-Dimensional Epithelium Equivalents.

The aim of this study is to investigate the repressive effects of enzyme-digested edible bird's nest (EBND) on the combination of arid environment and UV-induced intracellular oxidative stress, cell death, DNA double-strand breaks (DSBs) and inflammatory responses in human HaCaT keratinocytes and three-dimensional (3D) epithelium equivalents. An oxygen radical antioxidant capacity assay showed that EBND exhibited excellent peroxyl radical scavenging activity and significantly increased cellular antioxidant capacity in HaCaT cells. When EBND was administered to HaCaT cells and 3D epitheliums, it exhibited significant preventive effects on air-drying and UVA (Dry-UVA)-induced cell death and apoptosis. Dry-UVA markedly induced intracellular reactive oxygen species (ROS) generation in HaCaT cells and 3D epitheliums as quantified by CellROX® Green/Orange reagents. Once HaCaT cells and 3D epitheliums were pretreated with EBND, Dry-UVA-induced intracellular ROS were significantly reduced. The results from anti-γ-H2A.X antibody-based immunostaining showed that EBND significantly inhibited Dry-UVA-induced DSBs in HaCaT keratinocytes. Compared with sialic acid, EBND showed significantly better protection for both keratinocytes and 3D epitheliums against Dry-UVA-induced injuries. ELISA showed that EBND significantly suppressed UVB-induced IL-6 and TNF-α secretion. In conclusion, EBND could decrease arid environments and UV-induced harmful effects and inflammatory responses in human keratinocytes and 3D epithelium equivalents partially through its antioxidant capacity.

Enzyme-digested Colla Corii Asini (E'jiao) suppresses lipopolysaccharide-induced inflammatory changes in THP-1 macrophages and OP9 adipocytes.

Gut microbial lipopolysaccharides (LPS)-induced inflammatory responses in adipose tissue are associated with the dysfunction of adipocytes, insulin resistance and the development of metabolic syndrome. The aim of this study is to investigate (1) the effects of LPS on the differentiation and inflammatory responses of THP-1 monocytes and OP9 preadipocytes under serum free conditions and (2) the repressive effects of enzyme-digested Colla Corii Asini (CCAD) and fish gelatin (FGD) on LPS-induced inflammatory responses in THP-1 macrophages and OP9 adipocytes. Immunofluorescence and oil red O staining showed that a serum free medium supplied with phorbol 12-myristate 13-acetate (PMA) could induce differentiation and lipid accumulation in THP-1 cells as well as OP9 cells. ELISA showed that LPS significantly increased interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) secretions in PMA-differentiated THP-1 macrophages in a dose-dependent manner. LPS significantly suppressed lipid accumulation and adiponectin secretions, and enhanced IL-6 secretions in OP9 adipocytes. Both CCAD and FGD significantly reduced the levels of both macrophages- and adipocytes-derived inflammatory cytokines and increased the level of OP9-secreted adiponectin. In conclusion, LPS could induce inflammatory responses in both THP-1 and OP9 cells and cause dysfunction of OP9 adipocytes under the serum free conditions. CCAD and FGD can repress LPS-induced inflammatory responses in both THP-1 macrophages and OP9 adipocytes, and increase the secretion of adiponectin in OP9 adipocytes. They could be used as health care supplements for improving metabolic syndrome.

Local Irradiation of Mouse Tooth Germ Gives Insight into the Direct Effects of Irradiation on Root Development.

To elucidate the mechanism underlying the failure of root formation after irradiation, we established a method of local irradiation of the molar tooth germ and demonstrated that radiation directly affected dental root development. In the current study, to locally irradiate the lower first molars of 5-day-old C57BL/6J mice, we used lead glass containing a hole as a collimator. We confirmed that our local irradiation method targeted only the tooth germ. The irradiated root was immature in terms of apical growth, and dentin formation was irregular along the outside of the root apices. Moreover, calcified tissue apically surrounded Hertwig's epithelial root sheath, which disappeared abnormally early. This method using a local irradiation experimental model will facilitate research into radiation-induced disorders of dental root formation.

Peripheral-neuron-like properties of differentiated human dental pulp stem cells (hDPSCs).

Elucidating the mechanisms underlying human pain sensation requires the establishment of an in vitro model of pain reception comprising human cells expressing pain-sensing receptors and function properly as neurons. Human dental pulp stem cells (hDPSCs) are mesenchymal stem cells and a promising candidate for producing human neuronal cells, however, the functional properties of differentiated hDPSCs have not yet been fully characterized. In this study, we demonstrated neuronal differentiation of hDPSCs via both their expression of neuronal marker proteins and their neuronal function examined using Ca2+ imaging. Moreover, to confirm the ability of nociception, Ca2+ responses in differentiated hDPSCs were compared to those of rat dorsal root ganglion (DRG) neurons. Those cells showed similar responses to glutamate, ATP and agonists of transient receptor potential (TRP) channels. Since TRP channels are implicated in nociception, differentiated hDPSCs provide a useful in vitro model of human peripheral neuron response to stimuli interpreted as pain.

Hydrogen-Generating Silica Material Prevents UVA-ray-Induced Cellular Oxidative Stress, Cell Death, Collagen Loss and Melanogenesis in Human Cells and 3D Skin Equivalents.

Ultraviolet-A (UVA) irradiation induces harmful effects on skin cells and accelerates skin aging through oxidative stress. In this study, the effects of a hydrogen-generating silica material named ULH-002 against UVA injuries in human cells and 3D skin equivalents were investigated. The oxygen radical absorption capacity (ORAC) assay showed that both freshly prepared ULH-002 solutions and 7-day-old solutions exhibited equal peroxyl radical (ROO·) scavenging activities concentration-dependently. CellROX® green/orange staining showed that ULH-002 could reduce UVA-induced oxidative stress in human keratinocytes HaCaT and human gingival fibroblasts (HGFs). ULH-002 significantly prevented UVA-induced apoptotic/necrotic cell death and cell-viability decline in HGFs and keratinocytes, as shown by Annexin V/PI apoptosis assay and PrestoBlue assay, respectively. Immunostaining showed that ULH-002 prevented the UVA-induced deterioration of expression of both type IV and I collagens in the 3D skin equivalents, and similarly in monolayer HGFs. UVA-enhanced melanogenesis was observed in human melanocytes HMV-II and HMV-II cell-containing 3D skin equivalents, but markedly prevented by ULH-002 as demonstrated by Fontana-Masson's staining. In conclusion, our data suggested that ULH-002 could protect human keratinocytes and fibroblasts from UVA-induced injuries, prevent the loss of type IV and I collagens, as well as reduce melanogenesis. ULH-002 might be developed as a skin care reagent in the cosmetic industry.

Rapid differentiation of human dental pulp stem cells to neuron-like cells by high K+ stimulation.

As human-origin cells, human dental pulp stem cells (hDPSCs) are thought to be potentially useful for biological and medical experiments. They are easily obtained from lost primary teeth or extracted wisdom teeth, and they are mesenchymal stem cells that are known to differentiate into osteoblasts, chondrocytes, and adipocytes. Although hDPSCs originate from neural crest cells, it is difficult to induce hDPSCs to differentiate into neuron-like cells. To facilitate their differentiation into neuron-like cells, we evaluated various differentiation conditions. Activation of K+ channels is thought to regulate the intracellular Ca2+ concentration, allowing for manipulation of the cell cycle to induce the differentiation of hDPSCs. Therefore, in addition to a conventional neural cell differentiation protocol, we activated K+ channels in hDPSCs. Immunocyto-chemistry and real-time PCR revealed that applying a combination of 3 stimuli (high K+ solution, epigenetic reprogramming solution, and neural differentiation solution) to hDPSCs increased their expression of neuronal markers, such as ß3-tubulin, postsynaptic density protein 95, and nestin within 5 days, which led to their rapid differentiation into neuron-like cells. Our findings indicate that epigenetic reprogramming along with cell cycle regulation by stimulation with high K+ accelerated the differentiation of hDPSCs into neuron-like cells. Therefore, hDPSCs can be used in various ways as neuron-like cells by manipulating their cell cycle.

Type I collagen facilitates safe and reliable expansion of human dental pulp stem cells in xenogeneic serum-free culture.

BACKGROUND: Human dental pulp stem cells (DPSCs) are a readily accessible and promising cell source for regenerative medicine. We recently reported that a xenogeneic serum-free culture medium (XFM) is preferable to fetal bovine serum-containing culture medium for ex vivo expansion of DPSCs; however, we observed that, upon reaching overconfluence, XFM cells developed a multilayered structure and frequently underwent apoptotic death, resulting in reduced cell yield. Therefore, we focused on optimization of the XFM culture system to avoid the undesirable death of DPSCs. METHODS: We selected type I collagen (COL) as the optimal coating substrate for the cultureware and compared DPSCs cultured on COL in XFM (COL-XFM cells) to the conventional XFM cultures (XFM cells). RESULTS: Our results demonstrated that COL coating facilitated significantly higher rates of cell isolation and growth; upon reaching overconfluence, cell survival and sustained proliferative potential resulted in two-fold yield compared to the XFM cells. Surprisingly, after subculturing the overconfluent COL-XFM cultures, the cells retained stem cell behavior including stable cell growth, multidifferentiation potential, stem cell phenotype, and chromosomal stability, which was achieved through HIF-1α-dependent production and uniform distribution of collagen type I and its interactions with integrins α2ß1 and α11ß1 at overconfluency. In contrast, cells undergoing apoptotic death within overconfluent XFM cultures had disorganized mitochondria with membrane depolarization. CONCLUSION: The use of COL as a coating substrate promises safe and reliable handling of DPSCs in XFM culture, allowing translational stem cell medicine to achieve stable isolation, expansion, and banking of donor-derived stem cells.

Hmga2 regulation of tooth formation and association with Sox2 and Nanog expression.

Tooth formation is accomplished under strict genetic programs. Although patients with chromosome 12q14 aberration shows tooth phenotype including the size and eruption timing with bone growth anomaly, its etiology is uncertain. Here, we examined expression of Hmga2, which is encoded at chromosome 12q14, in mouse tooth germs and analyzed the involvement in lower first molar (M1) and mandibular bone development. Hmga2 expression was immunohistochemically detected at enamel organ and the surrounding mesenchyme of the M1 germs. The expression was dynamically changed with gestation and rapidly decreased in postnatal mice. In Hmga2-/- mice, the M1 germs and crowns were diminished in size, and formation and eruption of molars were delayed with mandibular bone growth retardation. Hmga2 cDNA or siRNA transfection showed that Hmga2 transcriptionally up-regulates expression of stem cell factors, Sox2 and Nanog. They were co-localized with Hmga2 in the germs, but differentially distributed at enamel organ and mesenchyme in Hmga2-/- mice. These results demonstrate that Hmga2 expressed in tooth germs regulates the growth, sizing and eruption and stem cell factor expression in different compartment of the germ and associates with mandibular bone growth. Although future studies are needed, the present study demonstrates HMGA2 regulation of tooth genesis with skeletal development.

Dental pulp cell bank as a possible future source of individual hepatocytes.

Mesenchymal stem cells (MSCs) as a source for regenerative medicine are now the subject of much clinical attention. There are high expectations due to their safety, low tumorigenic risk, and low ethical concerns. MSC therapy has been approved for acute graft-versus host diseases since 2015. Tooth-derived MSCs are known to have a great potential in their proliferation and differentiation capacities, even when compared with bone-marrow-derived MSCs. In particular, stem cells from human exfoliated deciduous teeth (SHEDs) are the best candidates for personal cell banking (dental pulp cell bank), because they can be obtained less invasively in the natural process of individual growth. SHEDs are known to differentiate into hepatocytes. There have been several studies showing the effectiveness of SHEDs on the treatment of liver failure in animal models. They may exert their effects either by repopulation of cells in injured liver or by paracrine mechanisms due to their immune-regulatory functions. Moreover, it may be possible to use each individuals' dental pulp cells as a future source of tailor-made differentiated hepatocytes in the context of a bioartificial liver or liver-on-a-chip to screen for drug toxicity.

Cellular responses of periodontal ligament stem cells to a novel synthesized form of calcium hydrogen phosphate with a hydroxyapatite-like surface for periodontal tissue engineering.

Calcium hydrogen phosphate with a hydroxyapatite-like surface (CHP-HA) is a novel synthesized compound designed to overcome the limitations of bioactive ceramics. It was originally applied as nano-sized HA strips covering core plates to enhance the degree of interfacial attachment. The objective of the present study was to examine the cellular attachment, proliferation, and osteogenic differentiation of periodontal ligament stem cells (PDLSCs) on a CHP-HA substrate in comparison with conventional nanohydroxyapatite (NanoHA). The PDLSCs were cultivated with either CHP-HA or NanoHA for cellular attachment, proliferation, and osteogenic differentiation assay. Osteogenic differentiation was examined using quantitative polymerase chain reaction and immunofluorescence after confirmation by Alizarin red staining. We found that between 14 and 21 days, CHP-HA exhibited a well-organized matrix distribution, a high degree of cell proliferation, and a high level of Alizarin red staining in comparison to NanoHA. Expression of all the osteogenic markers examined was increased significantly relative to NanoHA at 14 days, but no significant differences in some osteogenic genes were found at 21 days. Immunofluorescence revealed stronger staining in the CHP-HA group. In conclusion, PDLSCs cultivated with this novel CHP-HA show enhanced cellular responses. We propose that CHP-HA may be a promising alternative biomaterial for periodontal tissue engineering.

Establishment of xenogeneic serum-free culture methods for handling human dental pulp stem cells using clinically oriented in-vitro and in-vivo conditions.

BACKGROUND: Currently, ex-vivo handling of stem cells, including transport after harvest and therapeutic preparation, is generally done in culture media containing fetal bovine serum (FBS), which promotes cell attachment, proliferation, and differentiation. However, because of safety concerns associated with the use of FBS, including potential transmission of zoonotic agents and transplant rejection because of the incorporation of foreign proteins into the stem cells, there is a need for xenogeneic serum-free culture media for clinical handling of stem cells. METHODS: Dental pulp stem cells were derived from wisdom teeth donated by eight healthy volunteers and cultured in xenogeneic serum-free culture medium (XFM) or xenogeneic serum-containing culture medium (SCM). Cells were subjected to morphological, proliferation, karyotype, differentiation, marker expression, cryopreservation, and cytotoxic susceptibility analyses in vitro, as well as transplantation in vivo. RESULTS: In primary culture, XFM cells showed lower adhesion and slightly different morphology, although the single-cell size was similar to that of SCM cells. XFM cells exhibited typical mesenchymal stem cell (MSC) characteristics in vitro and in vivo, including marker gene/protein expression, trilineage differentiation potential, and hard, osteo-dentin tissue formation. Additionally, XFM cells maintained a normal karyotype in vitro and nontumorigenic potential in vivo; however, XFM cells were more susceptible to H2O2 and ultraviolet cytotoxic stimuli. XFM cells formed a multilayered structure showing excessive cell death/division in contrast to the monolayered structure of SCM cells when reaching overconfluence. Proliferation was disrupted in overconfluent XFM cells, and these cells could not be subcultured. Dimethyl sulfoxide-free cryopreserved XFM cells yielded similar results in all of the experiments. CONCLUSIONS: This study is the first reporting successful isolation and expansion of an MSC population from donor-derived tissue (dental pulp) under xenogeneic serum-free culture conditions, as well as the application of cryopreservation, using a research strategy based on clinically oriented in-vitro and in-vivo experiments.

Heterogeneous Human Periodontal Ligament-Committed Progenitor and Stem Cell Populations Exhibit a Unique Cementogenic Property Under In Vitro and In Vivo Conditions.

An undesirable complication that arises during dental treatments is external apical-root resorption, which causes root-cementum and root-dentin loss. To induce de novo cementogenesis, stem cell therapy is required. Cementum-forming cells (cementoblasts) are known to be differentiated from periodontal-lineage mesenchymal stem cells (MSCs), which are derived from the dental follicle (DF) in developing tissues and the periodontal ligament (PDL) in adult tissues, but the periodontal-lineage MSC type that is optimal for inducing de novo cementogenesis remains unidentified, as does the method to isolate these cells from harvested tissues. Thus, we investigated the cementogenic potential of DF- and PDL-derived MSCs that were isolated by using two widely used cell-isolation methods: enzymatic digestion and outgrowth (OG) methods. DF- and PDL-derived cells isolated by using both methods proliferated actively, and all four isolated cell types showed MSC gene/protein expression phenotype and ability to differentiate into adipogenic and chondrogenic lineages. Furthermore, cementogenic-potential analysis revealed that all cell types produced alizarin red S-positive mineralized materials in in vitro cultures. However, PDL-OG cells presented unique cementogenic features, such as nodular formation of mineralized deposits displaying a cellular intrinsic fiber cementum-like structure, as well as a higher expression of cementoblast-specific genes than in the other cell types. Moreover, in in vivo transplantation experiments, PDL-OG cells formed cellular cementum-like hard tissue containing embedded osteocalcin-positive cells, whereas the other cells formed acellular cementum-like materials. Given that the root-cementum defect is likely regenerated through cellular cementum deposition, PDL-OG cell-based therapies might potentially facilitate the de novo cellular cementogenesis required for regenerating the root defect.

Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine.

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.

Isolation and characterization of embryonic ameloblast lineage cells derived from tooth buds of fetal miniature swine.

Dental enamel formation, known as "amelogenesis," is initiated by cytodifferentiation of the ectodermally derived dental epithelium. Enamel cannot regenerate itself because once it is completely formed, ameloblasts are lost as the tooth erupts. Rodent teeth have been useful for studying the mechanisms of amelogenesis because ameloblast cell lines can be derived from the ever-growing incisors. However, higher mammals such as humans have no growing teeth, and cell lines derived from larger animals that are more similar to humans are required for higher fidelity studies. Here, we isolated embryonic enamel epithelium-derived epithelial cells from fetal swine. The explant culture of the developing deciduous molars that had been removed from the dental papilla-derived mesenchymal tissue and cells inside the tooth buds provided the epithelial cell population for the primary culture. To isolate the cell population, we performed a unique cell isolation technique called cell fishing. The isolated cells showed clear embryonic-stage ameloblast characteristics with appropriate gene/protein expressions of enamel matrix and proteinases, abundant glycogen pools, and secretory granular materials. They could be continuously subcultured several times and are presently being maintained. This cell population will facilitate the establishment of a stable cell line and allow us to characterize the definitive phenotype and functional behavior of porcine ameloblasts, which, in turn, promises to yield useful and practical findings that are more relevant than those provided by rodent studies. Finally, analysis of in vitro enamel formation will be important for engineering "bio-enamel" as a new dental therapy to restore enamel defects.

Establishment and characterization of a cell line designated Nur-1 derived from human endometrioid adenocarcinoma of uterine corps.

A new cell line designated Nur-1 has been established from human endometrioid adenocarcinoma, Grade 1, pT1a, PN1 (3/24), Stage IIIc (International Federation of Gynecology and Obstetrics/Union for International Cancer Control (FIGO/UICC TNM Classification of Malignant Tumours, 7th ed.). Cytological findings of Nur-1 cells reveal anaplastic and pleomorphic features such as anisonucleosis, nucleolar pleomorphism, and piling-up tendency in cellular arrangement. Distribution of the chromosome number is found at the hyperploid range, and the apparent marker chromosome has not been identified. The original tumor and graft of the Nur-1 cell line had a large amount of estrogen receptors and progesterone receptors, as revealed by immunohistochemistry. The cytokeratin pattern of the tumor was positive for cytokeratin-7 and negative for cytokeratin-20. However, a few cells were positive for cytokeratin-20 in the original tumor. Nur-1 cells express mRNA of estrogen receptors and progesterone receptors, cytokeratin-7, and cytokeratin-20 at 105 passages. These findings are consistent with the cytokeratin pattern of endometrial glandular cells. The cells make contact with each other via interdigitation and desmosomes. They possess bundles of microtubules and tonofilaments and many free ribosomes. Some cells have various sizes of phagosomes. The Nur-1 cell line exceeded 102 passages in 5 years, and multiplication of the cells is stable. The modal number of the Nur-1 cell line is 91-92 (56 %). The Nur-1 cells develop well-differentiated adenocarcinoma in tumors sustained in nude mice that resemble the original tumors.

Postnatal mandibular cheek tooth development in the miniature pig based on two-dimensional and three-dimensional X-ray analyses.

The miniature pig is a useful large laboratory animal model. Various tissues and organs of miniature pigs are similar to those of humans in terms of developmental, anatomical, immunological, and physiological characteristics. The oral and maxillofacial region of miniature pigs is often used in preclinical studies of regenerative dentistry. However, there is limited information on the dentition and tooth structure of miniature pigs. The purpose of this study was to examine the time-course changes of dentition and tooth structure (especially the root) of the miniature pig mandibular cheek teeth through X-ray analyses using soft X-ray for two-dimensional observations and micro-CT for three-dimensional observations. The mandibles of male Clawn strain miniature pigs (2 weeks and 3, 5, 7, 9, 11, 14, 17, and 29 months of age) were used. X-ray analysis of the dentition of miniature pig cheek teeth showed that the eruption pattern of the miniature pig is diphyodont and that the replacement pattern is vertical. Previous definitions of deciduous and permanent teeth often varied and there has been no consensus on the number of teeth (dentition); however, we found that three molars are present in the deciduous dentition and that four premolars and three molars are present in the permanent dentition. Furthermore, we confirmed the number of tooth roots and root canals. We believe that these findings will be highly useful in future studies using miniature pig teeth.

Establishment and characterization of METON myoepithelioma cell line derived from human palatal myoepithelioma: apical reference to the diverse differentiation potential.

Myoepithelioma is an extremely rare condition that accounts for 1-1.5 % of salivary gland tumors. It was formerly regarded as a subtype of pleomorphic adenoma, in which myoepithelial structural components predominated, but was listed as a separate disease entity in the 1991 World Health Organization classification (Seifert in Histological typing of salivary gland tumours. Springer, Berlin, 1991). Its histology is highly varied and recurrence is frequent (El-Naggar et al. in J Larygol Otol 103:1192-1197, 1989), with cases of malignant transformation having been reported (Seifert in Histological typing of salivary gland tumours. Springer, Berlin, 1991; Barnes et al. in Pathology and Genetics of head and neck tumours. IARC Press, Lyon, 2005), making this a difficult tumor to control in many cases. This is thought to be due to the multiple differentiation potential of myoepithelial cells, but the details are unknown. There have been a number of reports of the establishment of cell lines (Shirasuna et al. Cancer. 45:297-305, 1980; Jaeger et al. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 84:663-667, 1997), but numerous points remain unclear. We established a myoepithelial cell line designated METON, and investigated its characteristics. METON consists of cells with two different morphologies: spindle-shaped cells and epithelial-like cells. Then. we also used single-cell cloning method to establish various subclones (epithelial-like, spindle-like, and mixed epithelial-like/spindle-like cell lines). Among these, pluripotency markers were expressed by the mixed epithelial-like/spindle-like cell lines. The newly established cell line expressing these pluripotency markers will be extremely useful for elucidating the diverse histologies of salivary gland tumors.

Isolation and characterization of epithelial and myogenic cells by "fishing" for the morphologically distinct cell types in rat primary periodontal ligament cultures.

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.

Isolation and characterization of vascular endothelial cells derived from fetal tooth buds of miniature swine.

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.