Keishi Otsu

Concentration dependent inhibition of angiogenesis by mesenchymal stem cells

Mesenchymal stem cells (MSCs), which potentially transdifferentiate into multiple cell types, are increasingly reported to be beneficial in models of organ system injury. However, the molecular mechanisms underlying interactions between MSCs and host cells, in particular endothelial cells (ECs), remain unclear. We show here in a matrigel angiogenesis assay that MSCs are capable of inhibiting capillary growth. After addition of MSCs to EC-derived capillaries in matrigel at EC:MSC ratio of 1:1, MSCs migrated toward the capillaries, intercalated between ECs, established Cx43-based intercellular gap junctional communication (GJC) with ECs, and increased production of reactive oxygen species (ROS). These events led to EC apoptosis and capillary degeneration. In an in vivo tumor model, direct MSC inoculation into subcutaneous melanomas induced apoptosis and abrogated tumor growth. Thus, our findings show for the first time that at high numbers, MSCs are potentially cytotoxic and that when injected locally in tumor tissue they might be effective antiangiogenesis agents suitable for cancer therapy.

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.

Role of Epithelial-Stem Cell Interactions during Dental Cell Differentiation

Background: The role of dental epithelium in stem cell differentiation has not been clearly elucidated. Results: SP cells differentiated into odontoblasts by epithelial BMP4, whereas iPS cells differentiated into ameloblasts when cultured with dental epithelium. Conclusion: Stem cells can be induced to odontogenic cell fates when co-cultured with dental epithelium. Significance: This is the first report to show induction of ameloblasts from iPS cells. Epithelial-mesenchymal interactions regulate the growth and morphogenesis of ectodermal organs such as teeth. Dental pulp stem cells (DPSCs) are a part of dental mesenchyme, derived from the cranial neural crest, and differentiate into dentin forming odontoblasts. However, the interactions between DPSCs and epithelium have not been clearly elucidated. In this study, we established a mouse dental pulp stem cell line (SP) comprised of enriched side population cells that displayed a multipotent capacity to differentiate into odontogenic, osteogenic, adipogenic, and neurogenic cells. We also analyzed the interactions between SP cells and cells from the rat dental epithelial SF2 line. When cultured with SF2 cells, SP cells differentiated into odontoblasts that expressed dentin sialophosphoprotein. This differentiation was regulated by BMP2 and BMP4, and inhibited by the BMP antagonist Noggin. We also found that mouse iPS cells cultured with mitomycin C-treated SF2-24 cells displayed an epithelial cell-like morphology. Those cells expressed the epithelial cell markers p63 and cytokeratin-14, and the ameloblast markers ameloblastin and enamelin, whereas they did not express the endodermal cell marker Gata6 or mesodermal cell marker brachyury. This is the first report of differentiation of iPS cells into ameloblasts via interactions with dental epithelium. Co-culturing with dental epithelial cells appears to induce stem cell differentiation that favors an odontogenic cell fate, which may be a useful approach for tooth bioengineering strategies.

Stem cell sources for tooth regeneration: current status and future prospects

Stem cells are capable of renewing themselves through cell division and have the remarkable ability to differentiate into many different types of cells. They therefore have the potential to become a central tool in regenerative medicine. During the last decade, advances in tissue engineering and stem cell-based tooth regeneration have provided realistic and attractive means of replacing lost or damaged teeth. Investigation of embryonic and adult (tissue) stem cells as potential cell sources for tooth regeneration has led to many promising results. However, technical and ethical issues have hindered the availability of these cells for clinical application. The recent discovery of induced pluripotent stem (iPS) cells has provided the possibility to revolutionize the field of regenerative medicine (dentistry) by offering the option of autologous transplantation. In this article, we review the current progress in the field of stem cell-based tooth regeneration and discuss the possibility of using iPS cells for this purpose.

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. Hertwigs 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-β, 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.

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 β‐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. J. Cell. Physiol. 226: 2527–2534, 2011.

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 Hertwigs 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.

Cell dynamics in cervical loop epithelium during transition from crown to root: implications for Hertwig's epithelial root sheath formation

BACKGROUND AND OBJECTIVE Some clinical cases of hypoplastic tooth root are congenital. Because the formation of Hertwigs epithelial root sheath (HERS) is an important event for root development and growth, we have considered that understanding the HERS developmental mechanism contributes to elucidate the causal factors of the disease. To find integrant factors and phenomenon for HERS development and growth, we studied the proliferation and mobility of the cervical loop (CL). MATERIAL AND METHODS We observed the cell movement of CL by the DiI labeling and organ culture system. To examine cell proliferation, we carried out immunostaining of CL and HERS using anti-Ki67 antibody. Cell motility in CL was observed by tooth germ slice organ culture using green fluorescent protein mouse. We also examined the expression of paxillin associated with cell movement. RESULTS Imaging using DiI labeling showed that, at the apex of CL, the epithelium elongated in tandem with the growth of outer enamel epithelium (OEE). Cell proliferation assay using Ki67 immunostaining showed that OEE divided more actively than inner enamel epithelium (IEE) at the onset of HERS formation. Live imaging suggested that mobility of the OEE and cells in the apex of CL were more active than in IEE. The expression of paxillin was observed strongly in OEE and the apex of CL. CONCLUSION The more active growth and movement of OEE cells contributed to HERS formation after reduction of the growth of IEE. The expression pattern of paxillin was involved in the active movement of OEE and HERS. The results will contribute to understand the HERS formation mechanism and elucidate the cause of anomaly root.

Regulatory mechanisms of Hertwig׳s epithelial root sheath formation and anomaly correlated with root length

Teeth are composed of two domains, the enamel-covered crown and cementum-covered root. The mechanism for determining the transition from crown to root is important for understanding root anomaly diseases. Hertwig׳s epithelial root sheath (HERS) is derived from the dental epithelium and is known to drive the growth of root dentin and periodontal tissue. Some clinical cases of hypoplastic tooth root are caused by the cessation of HERS development. Understanding the mechanisms of HERS development will contribute to the study of the disease and dental regenerative medicine. However, the developmental biology of tooth root formation has not been fully studied, particularly regarding HERS formation. Here, we describe the mechanisms of HERS formation on the basis of analysis of cell dynamics using imaging and summarize how the growth factor and its receptor regulate cell behavior of the dental epithelium.

Sox2 contributes to tooth development via Wnt signaling

The transcription factor Sox2 is a stem cell marker that dictates cell lineage. It has been shown to mark the epithelial stem cells of the continuously growing mouse incisors. Sox2 also interferes with Wnt signaling by binding to β-catenin, a central mediator of the Wnt pathway. We show that these functions of Sox2 are essential for mouse molar development. Sox2 has previously been shown to play a role in the formation of new teeth from the existing dental epithelium. To assess Sox2 function related to cell migration within a tooth, we monitored cell movement by using a DiI system and observed that DiI moves from molar 1 to molar 2 during tooth development. However, upon temporal knockdown of Sox2, DiI remains in the molar 1 region. This study also provides novel insights into the role of Sox2 and the important validation of Sox2 as a potent target in Wnt signaling during tooth development. Our data reveal that the degradation of Wnt signaling caused by the knockdown of Sox2 results in a lack of cell migration during tooth development.