Functional role of Rho-kinase in ameloblast differentiation.

During tooth development, inner enamel epithelial (IEE) cells differentiate into enamel-secreting ameloblasts, a polarized and elongated cellular population. The molecular underpinnings of this morphogenesis and cytodifferentiation, however, are not well understood. Here, we show that Rho-associated coiled-coil-containing protein kinase (ROCK) regulates ameloblast differentiation and enamel formation. In mouse incisor organ cultures, inhibition of ROCK, hindered IEE cell elongation and disrupted polarization of differentiated ameloblasts. Expression of enamel matrix proteins, such as amelogenin and ameloblastin, and formation of the terminal band structure of actin and E-cadherin were also perturbed. Cultures of dental epithelial cells revealed that ROCK regulates cell morphology and cell adhesion through localization of actin bundles, E-cadherin, and ß-catenin to cell membranes. Moreover, inhibition of ROCK promoted cell proliferation. Small interfering RNA specific for ROCK1 and ROCK2 demonstrated that the ROCK isoforms performed complementary functions in the regulation of actin organization and E-cadherin-mediated cell-cell adhesion. Thus, our results have uncovered a novel role for ROCK in amelogenesis.

Establishment of Hertwig's epithelial root sheath cell line from cells involved in epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is an important event in the developmental process of various organs. In periodontal development during root formation of a tooth, this EMT has been a subject of controversy. Hertwig's epithelial root sheath (HERS), consisting of two epithelial layers, plays a role of inducing odontogenesis during root development and thereafter becomes fragmented. Some researchers have maintained that in the process of this fragmentation, some HERS cells change from epithelial to mesenchymal cells. Here, we established a HERS cell line (HERS01a) and examined its gene and protein expression. Immunohistochemical staining and real-time PCR analysis showed that HERS01a cells expressed vimentin and N-cadherin as mesenchymal markers as well as cytokeratin14, E-cadherin, and p63 as epithelial stem cell markers. In the presence of TGF-ß, HERS01a cells also expressed many more mesenchymal markers, as well as snail1 and 2 as EMT markers. Taken together, our data show that HERS01a displayed unique features associated with EMT in the root formation process, and will thus be useful for analyzing the biological characteristics of HERS and the molecular mechanism underlying the EMT.

Reduction of Egf signaling decides transition from crown to root in the development of mouse molars.

Mouse, rat, and human molars begin to form their roots after the completion of crown morphogenesis. Though several signaling pathways and transcription factors have been implicated in the regulation of molar crown development, relatively little is known about the regulatory mechanisms involved in the transition from crown to root development. Tooth root formation is initiated by the development of Hertwig's epithelial root sheath (HERS) from the cervical loop in the enamel organ. In this study we examined the change in epidermal growth factor (Egf) signaling during this transition process. Immunohistochemical studies showed that the expression of Egf receptors in the enamel organ disappear gradually in the process and are not observed in HERS. Here, to examine the effect of Egf on the transition, we used the organ culture method to examine the root development. In the presence of Egf, stellate reticulum (SR) cells between the inner and outer epithelial layers in the enamel organ actively proliferated and maintained the enamel organ, and the formation of HERS was not observed. On the other hand, in either the absence of Egf or the presence of the inhibitor of Egf receptors, the SR cells disappeared and HERS formation started. Subsequently, root formation proceeded in the culture period. Therefore, disappearance of SR area may be a key event that controls the timing of onset of HERS formation, and Egf may be one of regulatory factors involved in the change from cervical loop epithelium to HERS during root development.

Expression of osteogenic proteins during the intrasplenic transplantation of Meckel's chondrocytes: A histochemical and immunohistochemical study.

Meckel's chondrocytes, derived from the ectomesenchyme, have the potential to transform into other phenotypes. In this study, we transplanted cell pellets of Meckel's chondrocytes into isogenic mouse spleens and analyzed their phenotypic transformation into osteogenic cells using histological and immunohistochemical methods. With the increasing duration of transplantation, chondrocytes were incorporated into splenic tissues and formed a von Kossa-positive calcified matrix containing calcium and phosphoric acid, similar to that of intact bone. Type I, II, and X collagens, and the bone-marker proteins osteocalcin, osteopontin, osteonectin, and bone morphogenetic protein-2 (BMP-2) were immunolocalized in the matrix formed by the transplanted chondrocytes. Osteopontin and osteonectin were detected in the calcified matrix at earlier stages than osteocalcin and BMP-2. Type II collagen was expressed during the first week of transplantation, and type X collagen-positive cells appeared scattered during the initial stage of calcification, these collagens being later replaced by type I collagen formed by osteocyte-like cells. Electron microscopic observations revealed that chondrocytes surrounded by the calcified matrix transformed into spindle-shaped osteocytic cells accompanying the formation of bone-type thick-banded collagen fibrils. These results suggest that phenotypic switching of Meckel's chondrocytes can occur under in vivo conditions at a cellular morphological level.

Impaired vascular invasion of Cbfa1-deficient cartilage engrafted in the spleen.

Chondrocyte maturation and vascular invasion of cartilage are essential in the process of endochondral ossification. Cbfal-deficient (Cbfa1-/-) mice displayed a complete absence of osteoblast and osteoclast maturation as well as severely inhibited chondrocyte maturation in most parts of the skeleton. Although chondrocyte maturation and mineralization were observed in restricted areas of Cbfa1-/- mouse skeleton, vascular invasion of calcified cartilage was never noted. To investigate the possibility of chondrocyte maturation and vascular invasion in Cbfal-/- cartilage and the role of the hematopoietic system in the process of vascular invasion, we transplanted embryonic day 18.5 (E18.5) Cbfa1-/- femurs, which are composed of immature chondrocytes, into spleens of normal mice. One week later, the transplanted femurs contained terminally differentiated chondrocytes expressing osteopontin, bone sialoprotein (BSP), and matrix metalloproteinase (MMP) 13. In the diaphyses of the transplants, the cartilage matrix was mineralized and the cartilage was invaded by vascular vessels and osteoclasts. However, chondrocyte maturation and vascular invasion were severely retarded in comparison with transplants of E14.5 wild-type femurs, in which the cartilage was rapidly replaced by bone, and neither mature osteoblasts nor bone formation were observed. In primary culture of Cbfa1-/- chondrocytes, transforming growth factor (TGF) beta1, platelet-derived growth factor (PDGF), interleukin (IL)-1beta, and thyroid hormone (T3) induced osteopontin and MMP-13 expression. These findings indicated that factors in the hematopoietic system are able to support vascular invasion of cartilage independent of Cbfal but are less effective without it, suggesting that Cbfal functions in cooperation with factors from bone marrow in the process of growth plate vascularization.

Phenotypic switching of in vitro mandibular condylar cartilage during matrix mineralization.

In order to analyze the phenotypic conversion of chondrocytes, mandibular condyles of mice and rabbits were cultured under cell and organ culture systems, and then examined by a combination of morphological and biochemical procedures. In organ culture, mandibular condylar cartilage (MCC) obtained from newborn mice began to mineralize from the central zone and then progressively widened towards the peripheral zone. Electron microscopic observations showed that with the increasing duration of the organ culture, chondrocytes at the central zone converted into spindle-shaped osteoblastic cells accompanying the formation of the bone type of thick-banded collagen fibrils. To obtain a better understanding of the chondrocytic conversion, immunolocalizations for type I and type X collagens and osteocalcin (OC) were examined in mouse MCC cells in cell culture. Type X collagen and OC were expressed almost simultaneously at the late stage of culture, and type I collagen was detected along the calcified nodules after the production of these proteins. Northern blot analysis in cell cultures of rabbit MCC indicated that type II collagen and alkaline phosphatase (ALPase) messenger ribonucleic acids (mRNAs) were highly expressed at day 7, but subsequently decreased. In contrast, mRNA for type I collagen was expressed at a low level on day 7 and peaked on day 12. The present results suggest that, morphologically and biochemically, cellular modification in MCC cells under culture conditions occurs at a cellular morphological level and also at marker-gene-expression level.

Imaging analysis of osteogenic transformation of Meckel's chondrocytes from green fluorescent protein-transgenic mice during intrasplenic transplantation.

Our previous studies demonstrated that Meckel's chondrocytes, which are derived from ectomesenchyme, have the potential to transform into osteogenic phenotypes. The present study aimed to clarify the role of cell origin in the phenotypic transformation of chondrocytes. Cell pellets from ectomesenchyme-derived Meckel's cartilage and mesoderm-derived costal cartilage from green fluorescent protein (GFP)-transgenic mice were transplanted into the spleen for up to 4 weeks. Chondrocyte pellets from both cartilages adapted well to the splenic tissues and formed an alizarin red-positive calcified matrix, with increasing duration of transplantation. Following the production of cartilage-specific type II and type X collagens, newly-formed type I collagen appeared in the chondrocyte pellets from Meckel's cartilage during the late stage of transplantation. Although the bone-marker proteins: osteocalcin, osteopontin, osteonectin and bone morphogenetic protein-2, were detected in pellets from both Meckel's and costal cartilage, only type I collagen in Meckel's cartilage was a significant marker protein for detecting transformation. These bone-type protein-producing cells represented osteogenic cells transformed from GFP-expressing cells, rather than from recipient cells. These results indicate that neural crest-derived Meckel's cartilage displays a higher potential for phenotypic switching than mesoderm-derived costal chondrocytes under in vivo conditions.

Differentiation of induced pluripotent stem cells into dental mesenchymal cells.

Similar to embryonic stem cells, induced pluripotent stem (iPS) cells can differentiate into various cell types upon appropriate induction, and thus, may be valuable cell sources for regenerative medicine. However, iPS cells have not been reported to differentiate into odontogenic cells for tooth regeneration. Here we demonstrated that neural crest-like cells (NCLC) derived from mouse iPS cells have the potential to differentiate into odontogenic mesenchymal cells. We developed an efficient culture protocol to induce the differentiation of mouse iPS cells into NCLC. We confirmed that the cells exhibited neural crest (NC) cell markers as evidenced by immunocytochemistry, flow cytometry, and real-time reverse transcription-polymerase chain reaction. Further, in recombination cultures of NCLC and mouse dental epithelium, NCLC exhibited a gene expression pattern involving dental mesenchymal cells. Some NCLC also expressed dentin sialoprotein. Conditioned medium of mouse dental epithelium cultures further enhanced the differentiation of NCLC into odontoblasts. These results suggest that iPS cells are useful cell sources for tooth regeneration and tooth development studies.

In vitro adipocytic conversion in Meckel's chondrocytes in response to a fatty acid-containing medium.

Chick serum (CKS) contains factors that stimulate adipocytes in Meckel's chondrocytes in vitro. In the present study, we analyzed levels of fatty acids in CKS, and further examined whether these had the potential to convert chondrocytes to adipocytes. Phenotypic changes were evaluated by light and electron microscopies, bromodeoxyuridine (BrdU) incorporation, triglyceride assays, and immunocytochemistry. We showed that CKS contained high levels of fatty acids, and a mixed medium containing 5 particular fatty acids inhibited DNA synthesis and the proliferation of chondrocytes as it facilitated their differentiation into adipocytes. The adipocytes produced were sudan-positive multilocular cells that morphologically and histochemically resembled adipocytes induced by the CKS-containing medium. Almost all lipid droplet-containing cells were positive for leptin and alpha-glycerophosphate dehydrogenase (GPDH), as evaluated by immunoperoxidase staining, and their triglyceride concentrations markedly increased during 4 to 6 days of culture. These results suggested that specific fatty acids in CKS are involved in the adipocytic conversion of Meckel's chondrocytes.

Insulin-like growth factor-I stimulates cell proliferation in the outer layer of Hertwig's epithelial root sheath and elongation of the tooth root in mouse molars in vitro.

To elucidate the mechanism of root formation in tooth development, we examined the role of insulin-like growth factor I (IGF-I) on early root formation in mandibular first molar teeth from 5-day-old mice. Immunohistochemistry revealed the specific localization of the IGF-I receptor in Hertwig's epithelial root sheath (HERS) in the tooth root. The effect of IGF-I on root development, especially on HERS, was subsequently examined in vitro. The control culture showed normal development of HERS and the periodontium, resembling that in vivo. However, the presence of 100 ng/ml IGF-I resulted in elongation of HERS and increased cell proliferation in its outer layer. These effects were negated by the addition of antibodies specific for IGF-I. Thus, we propose that IGF-I is involved in early root formation by regulating the mitotic activity in the outer layer of HERS.

Induction of adipogenesis by the intrasplenic transplantation of chick serum clots.

Chick serum contains a factor that stimulates adipogenesis in Meckel's chondrocytes in vitro. The present study examined whether chick serum has a capacity for adipogenic induction in vivo, by transplanting serum clots (created by drying chick serum for up to 4 weeks) into mouse spleens. Specimens were harvested for histological analyses, which included light and electron microscopy and immunohistochemistry. The transplanted serum clots induced the appearance of lipid droplet-containing cells in splenic cords and sinus. Almost all the lipid droplet-containing cells were positive for sudan staining and consisted of multilocular lipid vacuoles. Immunostaining showed that the adipocytes induced by transplantation of the serum clots initially appeared as peroxisome proliferator-activated receptor-gamma (PPARgamma)-positive cells and developed into leptin and alpha-glycerophosphate dehydrogenase (GPDH)-producing cells, in addition to type III collagen synthesis. Furthermore, double immunofluorescence staining revealed that the immunoreactivity for GPDH was detected not only in stromal cells but also in macrophages. It was thus confirmed that stromal cells and macrophages in the spleen contain lipid droplets as seen in intact white adipose cells. The present results suggest that chick serum contains factors for adipocyte induction not only in vitro but also in vivo, and that the adipogenic potential does not depend on the supplements used during the cell culture.

Phenotypic characteristics of adipocytes generated from Meckel's chondrocytes in response to chick serum in vitro.

When present in the culture medium, chick serum (CKS) modulated the phenotypic change from chondrocytes of Meckel's cartilage to adipocytes in vitro, as revealed by light and electron microscopy, the incorporation of BrdU, and immunocytochemistry. CKS inhibited DNA synthesis in chondrocytes and the proliferation of these cells, while it facilitated the differentiation to adipocytes. CKS contributed to phenotypic changes in undifferentiated chondrocytes, but did not affect the characteristics of differentiated chondrocytes. Electron microscopy revealed that the lipid droplets in adipocytes were enclosed by limiting membranes that fused to yield larger lipid droplets. Immunocytochemical staining of adipocytes with stage-specific antibodies revealed the presence of immunoreactive uncoupling protein (UCP-1) and peroxisome proliferator-activated receptor (PPARgamma) in immature adipocytes, and leptin and glucose transporter (Glut-4) in mature adipocytes. The adipocytes that were formed in the present study were multilocular adipocytes that contained many small lipid droplets, but in many ways they resembled white adipocytes. CKS contains a high level of estrogen, compared with fetal bovine serum, and it is possible that estrogen might have induced the differentiation to adipocytes.