Research projects

Mesenchymal stem cells (MSCs) are known for their multipotency and are characterized by their ability to differentiate into adipocytes, chondrocytes, osteocytes, and fibroblasts. In vivo, MSCs have been suggested to function as supportive cells for hematopoietic stem cells and to be involved in tissue fibrosis. This study aimed to demonstrate that MSCs can differentiate into CD45-positive macrophages both in vivo and in vitro, thereby contributing to immune responses. Surprisingly, we found that when freshly isolated mouse MSCs were reintroduced into mice, a subset of these cells became CD45-positive. Furthermore, we discovered that even in culture, under conditions promoting macrophage differentiation, MSCs could give rise to CD45-positive macrophages.

我々は、マウス骨髄移植モデルにて、骨髄移植時にCD45陰性であったGFP標識新鮮間葉系幹細胞（MSC）が、骨髄移植後移植後にCD45陽性となったことを突き止めた。これらの細胞はMHC Class II陽性、かつCollagen I陽性であることから、いわゆるFibrocyte、あるいはマクロファージの特徴を備えた細胞であることが示唆された。そこで、新鮮間葉系幹細胞（MSC）はFibrocyteを経て線維芽細胞に分化する他に、MHC Class II陽性の抗原提示細胞に分化する可能性を示すため、昨年度は骨髄より分離したMSCを本来は造血幹細胞由来とされている樹状細胞やマクロファールなどの抗原提示細胞に分化するかを確認した。その結果、骨髄の新鮮MSCがCD45陽性細胞に分化することをin vitroで確認した。CD45陽性細胞はCD68とCD206も陽性であることも確認できており、マクロファージであることをほぼ同定した。さらに本年度は新鮮間葉系幹細胞をマクロファージに分化誘導した後に、Single Cell RNA解析を実施した。その結果、マクロファージマーカーを発現する細胞には２つの群が存在することが明らかになった。最終年度ではこの２群の細胞について詳細に検討をする。

This study aimed to develop regenerative medicine cell products by isolating mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) from extracted teeth, which are medical waste. Initially, we attempted to isolate stem cells from dental pulp tissue using CD271Low+CD90High+ and CD73+ as markers, but the cell yield from a single tooth was insufficient. Therefore, we focused on surplus connective tissue fragments from secondary oral implant surgeries. We established physical and chemical processing protocols for minute tissues and constructed a stable MSC supply system through adherent culture separation methods. Regarding HSCs, we discovered they were virtually absent in dental pulp tissue, leading us to shift our approach to isolation from surplus bone tissue obtained during orthognathic surgery. This research established a foundation for developing sustainable stem cell therapeutic products using surplus oral tissue specimens.

In this study, we concentrated on human dental pulp tissue-derived LNGFRLow+THY-1High+ hDPSCs (human Dental Pulp Stem Cells; hDPSCs). The results of culturing the cells in Xenofree Mesenchymal Stem Cell Medium showed that the cell proliferation ability of the hDPSCs was similar to that of the conventional medium. The results of this experiment suggest that (1) the problem of heterogeneity of conventional cells can be overcome by uniform separation of stem cells using specific cell surface markers and (2) "xeno-free culture" without "serum components of foreign animal origin" is possible. We believe that this research project has expanded the possibility of clinical application of hDPSCs.