| Project/Area Number |
15390452
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Orthopaedic surgery
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| Research Institution | The University of Tokyo |
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Principal Investigator |
CHUNG Ung-il The University of Tokyo, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (30345053)
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| Co-Investigator(Kenkyū-buntansha) |
KAWAGUCHI Hiroshi The University of Tokyo, Hospital, Associate Professor, 医学部附属病院, 助教授 (40282660)
NAKAMURA Kouzou The University of Tokyo, Hospital, Professor, 医学部附属病院, 教授 (60126133)
IKEDA Toshiyuki The University of Tokyo, Hospital, Visiting Assistant Professor, 医学部附属病院, 客員助手 (80322759)
筑田 博隆 東京大学, 医学部附属病院, 助手 (30345219)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2005: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥7,700,000 (Direct Cost: ¥7,700,000)
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| Keywords | embryonic stem (ES) cells / osteoblast differentiation / chondrocyte differentiation / optimal differentiation condition / culture methods / regenerative medicine / 骨 / 軟骨 / 再生 / 胚性幹細胞 / 分化誘導 / 幹細胞 / 胚性幹細胞(ES) |
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| Research Abstract |
Mice expressing GFP transgenes under the control of promoter fragments of rat type I collagen gene and type U collagen gene were mated with wild-type mice, blastocytes were obtained and embryonic stem (ES) cells were isolated. After retroviruses expressing these transgenes were constructed, wild-type ES cells were infected with them and cell lines were established. ES cells from these mice, which fluoresce solely upon osteoblast or chondrocyte differentiation, were utilized as cell sensors for screening differentiation-inducing factors. We constructed adenoviruses expressing BMP receptors, Hh signaling molecule, Smo, Wnt transcription factor, LEF-1 and Runx2. Then, by exhaustively combining these osteoblast-differentiation inducing factors with above-mentioned ES cell sensor, we found that combination of Runx2 and BMP most strongly induced osteoblast differentiation.
As for cartilage-differentiation inducing factors, BMP and Sox signaling pathways were screened with the ES cell sensor a
… More
nd we found that combination of Sox9 and Sox5 or Sox6 most strongly induced cartilage differentiation.
Through these experiments, we optimized signaling for osteoblast or chondrocyte differentiation.
We also determined the optimal condition for proliferation of the ES cell lines in monolayer culture without differentiation (passaging intervals and best colony size). Aiming to use human ES cells in the near future, we purchased cynomolgus embryonic stem cells and gained experience in cultivating and differentiating them. We planted osteoblast-like and chondrocyte-like cells differentiated in the above determined optimal condition and cultured them on atelocollagen film. We succeeded in constructing sufficient matrix. We confirmed matrix calcification and metachromasie by toluidine blue staining. We transplanted these sheets to mouse bone and cartilage defects. One-four months after transplantation, we euthanized them after anesthesia and made histological and immunohistological examinations of the transplanted parts, through which we observed good regeneration in the bone-defect model but no significant hearing effects in the cartilage-defect model. We speculate that cartilage differentiation signal is not fully optimized. We plan to continue screening, considering various combinations with other signaling pathways. Less
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