Research projects

In human and mouse cancer tissues, we succeeded in isolating and analysis without culture operation around stromal cells and cancer epithelial cells. This is useful as a method of analyzing surface markers to separate the prospective mesenchymal stem cell

Homeostasis of periodontal tissue have been made by local periodontal ligament cells and alveolar bone cells. It has been reported that stem cell-like cells from the trunk migrate to periodontal tissue, and contributes to the regeneration and repair of periodontal tissue. Furthermore, these cells express the specific marker (PDGFRa) of bone marrow mesenchymal stem cells. Mesenchymal stem cells present in the periodontal tissue. The developmental origin of periodontal tissue is neural crest cells. We tried to induce neural crest cells from human iPS cells. It is the goal of this study aims to connect to periodontal tissue regeneration therapy , and obtained the following results. (1) We succeeded establishment of a method to generate human iPS cells derived human neural crest cells. (2) We conformed induced neural crest cells differentiated into osteo, chondro, fat, and peripheral nerve cells. (3) We verified that induced neural crest cells have highest migration potency.

（1）For the purpose of actual treatment, We have become possible to use human therapeutic iPS cells . （2）In the future, We suppose the use of human therapeutic iPS cells . In practical application, it is very important problem to use the conventional culture system. Because these system use fetal bovine serum and non human feeder cell to culture of human iPS cells. To overcome these problems, we established non serum / non feeder culture system of human therapeutic iPS cells . We think these steps (feeder free / xeno free culture system)are very notable in this study. （3）We succeeded inducing neural crest like cells from human iPS cells. Neural crest is developmental origin of periodontal tissue.

We evaluated the usefulness of 3D cultured human dental pulp stem cells (3D hDPSCs) in preventing osteonecrosis of the jaw by transplanting them into the tooth extraction sockets of immunodeficient mice. Transplantation of 2D cultured human dental pulp stem cells (2D hDPSCs) was unable to prevent osteonecrosis of the jaw, but transplantation of 3D hDPSCs covered the tooth extraction socket with epithelium and no bone exposure was observed. Compared to the group transplanted with 2D hDPSCs, the group transplanted with 3D hDPSCs showed bone formation and the rate of empty lacunae was significantly lower. In addition, it was confirmed that 3D hDPSCs expressed high levels of FGF2. These results suggest that FGF2 expressed in 3D hDPSCs contributes to the prevention of osteonecrosis of the jaw.

本研究では、口腔内マイクロバイオーム由来の炎症誘導性細菌の同定と制御性T細胞（regulatory T cell, Treg）の機能解析を行い消化器癌の進展における関与を解明することを目的とする。 歯周病モデルの作製について、本研究では、C3H/HeNJclおよびDBA/2JJC1実験マウスを用いて縫合糸を用いた歯頸部結紮法により歯周病感染を誘導する実験を行った。歯周病モデルにおける腫瘍の生着と増殖の検証について、歯頸部結紮法により歯周病を誘導したC3H/HeNJclおよびDBA/2JJC1マウスを用いてマウス由来の腫瘍細胞であるLM8およびKLN205をそれぞれ背部皮下に105cells移植を行い腫瘍細胞の生着と増殖の観察を行った。腫瘍細胞の移植４週間後に背部に形成された腫瘍組織の重量を計測した結果、歯周病群において腫瘍形成が亢進している傾向が確認された。 次に、潰瘍性大腸炎関連大腸癌と歯周病の関連については、潰瘍性大腸炎関連大腸癌（UC関連大腸癌）は大腸での炎症を背景として発生する癌である。今回、UCマウスモデルとして良く用いられているデキストラン硫酸ナトリウム（DSS）誘発性炎症性腸疾患モデルを用いて、歯周病が腸内の炎症に及ぼす影響を検討した。その結果、歯周病を起こしたマウスではDSSによる炎症がより強く起こることが分かった。このことは、歯周病が腸炎を増悪させ、UC関連癌発生リスクを上昇させる可能性を示唆した。今後は炎症を惹起させる原因となっている免疫細胞に着目し、さらなるメカニズム解明に繋げる予定である。

We clarified that the appearance of CD44 was not only a cancer stem cell surface marker but also functionally important in the oral squamous cell carcinoma. Especially, we thought to be useful as the cancer stem cell marker that the expression of variant is related to the metastasis.

We performed a study of the results of gene panel testing performed on specimens of oral squamous cell carcinoma operated on in our department and the differences in histopathology, and found a correlation between the number of Actionable mutations and grade of malignancy. In other words, the higher the number of Actionable mutations, the higher the grade of WPOI classification, which indicates clinicopathologic malignancy.

Our group focused on cancer-associated mesenchymal / stromal cells of cetuximab-resistant oral squamous cell carcinoma. We aimed at personalized treatment by constructing expression profiles of CD44v and novel mesenchymal markers.Analysis of cetuximab-resistant undifferentiated oral squamous cell carcinoma using tissue sample revealed that tissue-derived cells expressed both epithelial marker, EpCAM and mesenchymal marker, PDGFRα.The cancer stem cell marker CD44v-positive cells were identified in both cell fractions.

In this study, we realized the induction of neural crest cells, which was feeder-free, xeno-free iPS culture method. It was confirmed that neural crest cells existed in the periodontal ligament tissue of the mouse and some of them expressed PDGFRα and CD44 which are mesenchymal stem cell markers. This suggested that neural crest derived mesenchymal stem cells may be a potential source of cells for periodontal tissue regeneration therapy. In order to transplant these cells into real periodontal disease model mice, the disease model mice were prepared and evaluated. Periodontal disease induction model induced bone destruction by ligating 5-0 silk thread to mouse molar tooth. To confirm the technical reproducibility. Now, we have been preparing for transplantation.

The present study aims to induce neural crest cells from iPS cells and restore function of damaged nerve tissue. We established iPS cell culture method with feeder-free, xeno-free system for inducing neural crest cells. In order to investigate whether the induced neural crest cell population actually has the potential to restore peripheral nerve injury, we started the creation of the sciatic nerve cutting model and its functional evaluation. We found that transplanted induced neural crest cells possess the properties of mesenchymal stem cells and vascular endothelial cells that could also be induced into regenerating nerve tissues. These cells expressed peripheral nerve markers in transplantation tissues.

It has been advocated that Wnt, Notch signals involve molecular mechanism of self-renewal or differentiation in various somatic stem cells. In mammalian development, stem cell controls complicated organizational structure as high-order function, while controlling self-renew and differentiation. In this project, we attempted to clarify self-renewal and cell lineage decision mechanism of MSC. Our method was to visualize Wnt, Notch signals, which has a potential for temporospatial controls of stem cells. By visualizing Wnt, Notch signals, we could analyze how Wnt/Notch separates their roles, or interacts with other molecules in order for self-replication.

In order to establish an in vivo evaluation system for quantitative bone formation, we worked on the establishment of models to evaluate quantitative bone formation in mice and rats. In order to establish an evaluation system for new bone replacement materials, we are now histochemically examining the autogenous bone replacement rate using clinically used (approved) Biomaterials, b-TCP, and carbonate apatite. We continue to develop new materials for bone disease treatment that have "antibacterial properties," "a shape with excellent clinical usability," "osteoinductive ability to achieve a high autogenous bone replacement rate," and "intrinsic mesenchymal stem cell accumulation function.

Aiming to develop antibacterial implants effective against the bacterial causes of peri-implantitis, which hinders the success of dental implant treatment. We created an antibacterial implant by fixing silver ions onto titanium surfaces using a special method and confirmed its antibacterial effect against the main causative bacteria in bacterial culture experiments. Furthermore, in a peri-implantitis model experiment using mice, we demonstrated that this antibacterial implant significantly suppressed bone resorption and inflammation caused by peri-implantitis. These results could contribute to improving the long-term outcomes of prevention and treatment of peri-implantitis.

The purpose of this study was to investigate the efficacy of dental pulp stem cell transplantation for the prevention and treatment of bisphosphonate-related osteonecrosis of the jaw. We previously identified isolation markers of human dental pulp stem cells and reported that purified dental pulp stem cells by flow cytometry show high proliferation and osteogenic potential. In the present study, it was suggested that transplantation of purified dental pulp stem cells into immunodeficient mice after taking bisphosphonate and tooth extraction has the ability to prevent osteonecrosis of the jaw.

We confirmed that prospective isolatedMSCs showed a 120, 000-fold higher colony forming frequency thanthe conventional MSCs. Furthermore, Our results suggested that MSCs in adultbone marrow have at least two developmental origins, one of which is the neural crest. Furthermore, prospectively enrichedMSCs are a promising candidate for the efficient generation of high-quality iPS cells. This photocurable solution also has adequate viscosity at room temperature before curing, and it manipulation is enhanced. It is assumed that forms suitable for various bone defects will thus be easily rendered, and it is also assumed that the shape can be retained with the mechanical strength required by photocurable.