研究者詳細 - 寳田　剛志

論文 - 寳田　剛志

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Development of cartilage tissue using a stirred bioreactor and human iPSC-derived limb bud mesenchymal cells. 国際誌

Yuki Fujisawa, Tomoka Takao, Daisuke Yamada, Takeshi Takarada

Biochemical and biophysical research communications 687 149146 - 149146 2023年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

Production of cartilaginous particles for regenerative medicine requires a large supply of chondrocytes and development of suitable production techniques. Previously, we successfully produced human induced pluripotent stem cell (hiPSC)-derived limb bud mesenchymal cells (ExpLBM cells) with a high chondrogenic differentiation potential that stably proliferate. It may be possible to use these cells in combination with a stirred bioreactor to develop a tissue-engineered cell culture technology with potential for scale-up to facilitate production of large amounts of cartilaginous particles. ExpLBM cells derived from 414C2 and Ff-I 14s04 (human leukocyte antigen homozygous) hiPSCs were seeded into a stirred bioreactor containing cartilage induction medium. To characterize the cartilaginous particles produced, we performed real-time quantitative reverse transcription-polymerase chain reaction and histological analyses. Additionally, we transplanted the cartilage tissue into osteochondral defects of immunocompromised rats to assess its functionality, and evaluated engraftment of the grafted tissue. We successfully produced large amounts of cartilaginous particles via cartilage induction culture in a stirred bioreactor. This tissue exhibited significantly increased expression levels of type II collagen (COL2), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9), as well as positive Safranin O and Toluidine blue staining, indicating that it possesses characteristics of hyaline cartilage. Furthermore, engrafted tissues in osteochondral knee defects of immunodeficient rats were positively stained for human vimentin, COL2, and ACAN as well as with Safranin O. In this study, we successfully generated large amounts of hiPSC-derived cartilaginous particles using a combination of tissue engineering techniques. This method is promising as a cartilage regeneration technology with potential for scale-up.

DOI： 10.1016/j.bbrc.2023.149146

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Fabrication of shape-designable cartilage from human induced pluripotent stem cell-derived chondroprogenitors using a cell self-aggregation technique. 国際誌

Tomoyuki Ota, Tomoka Takao, Ryosuke Iwai, Takeshi Moriwaki, Yohei Kitaguchi, Yuki Fujisawa, Daisuke Yamada, Yoshihiro Kimata, Takeshi Takarada

Biomedical materials (Bristol, England) 2023年10月

記述言語：英語掲載種別：研究論文（学術雑誌）

With the advancement of tissue engineering technologies, implantable materials have been developed for use in facial plastic surgery, including auriculoplasty and rhinoplasty. Tissue-engineered cartilage comprising only cells and cell-produced extracellular matrix is considered valuable as there is no need to consider problems associated with the absorption of scaffold materials and the generation of immune responses. However, it is exceedingly difficult to produce large-sized complex shapes of cartilage without the use of scaffolds. In this study, we describe the production of shape-designable cartilage using a novel cell self-aggregation technique (CAT) and chondroprogenitor cells derived from human induced pluripotent stem cells as the source. The method described does not require special equipment such as bio-3D printers, and the produced tissue can be induced into well-matured cartilage with abundant cartilage matrix in vitro. Using CAT, we were able to generate cartilage in the form of rings or tubes with adjustable inner diameter and curvature, over a range of several centimeters, without the use of scaffolds. The in vitro fabrication of shape-designable cartilage using CAT will undoubtedly contribute to developments in the field of facial plastic surgery.&#xD.

DOI： 10.1088/1748-605X/ad02d1

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ヒトiPS細胞由来肢芽間葉系細胞の発生機序の検討

高尾知佳, 大曽根達則, 山田大祐, 中田英二, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 97 ( 8 ) S1599 - S1599 2023年8月

ヒトiPS細胞由来肢芽間葉系細胞を用いた骨再建法の開発

佐藤浩平, 高尾知佳, 中田英二, 藤澤佑樹, 山田大祐, 上原健敬, 藤原智洋, 依光正則, 国定俊之, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 97 ( 8 ) S1868 - S1868 2023年8月

悪性末梢神経鞘腫瘍におけるPRRX1の悪性化因子としての役割

たき平将太, 中田英二, 板野拓人, 片山晴喜, 藤原智洋, 国定俊之, 山田大祐, 高尾知佳, 宝田剛志, 尾崎敏文

日本整形外科学会雑誌 97 ( 8 ) S1890 - S1890 2023年8月

ヒトiPS細胞由来肢芽間葉系細胞の発生機序の検討

高尾知佳, 大曽根達則, 山田大祐, 中田英二, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 97 ( 8 ) S1599 - S1599 2023年8月

ヒトiPS細胞由来肢芽間葉系細胞を用いた骨再建法の開発

佐藤浩平, 高尾知佳, 中田英二, 藤澤佑樹, 山田大祐, 上原健敬, 藤原智洋, 依光正則, 国定俊之, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 97 ( 8 ) S1868 - S1868 2023年8月

悪性末梢神経鞘腫瘍における治療標的PRRX1の同定と新規創薬開発の可能性

たき平将太, 山田大祐, 岡本真幸, 高尾知佳, 中田英二, 板野拓人, 藤原智洋, 国定俊之, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 97 ( 6 ) S1428 - S1428 2023年6月

ヒト多能性幹細胞から誘導した肢芽間葉系細胞と、その拡大培養法の開発

中田英二, 山田大祐, 高尾知佳, たき平将太, 藤原智洋, 尾崎敏文, 宝田剛志

移植 57 ( 4 ) 363 - 363 2023年4月

ヒトiPS細胞から誘導した肢芽間葉系細胞による軟骨シートの作製

藤澤佑樹, 高尾知佳, 佐藤正人, 豊田恵利子, 山田大祐, 中田英二, 尾崎敏文, 宝田剛志

移植 57 ( 4 ) 362 - 362 2023年4月

悪性末梢神経鞘腫瘍におけるPRRX1の治療標的分子としての可能性

たき平将太, 中田英二, 大曽根達則, 山田大祐, 高尾知佳, 佐藤浩平, 畑利彰, 藤原智洋, 国定俊之, 宝田剛志, 尾崎敏文

日本整形外科学会雑誌 97 ( 3 ) S1040 - S1040 2023年3月

A novel chondrocyte sheet fabrication using human-induced pluripotent stem cell-derived expandable limb-bud mesenchymal cells. 国際誌

Tomoka Takao, Masato Sato, Yuki Fujisawa, Eriko Toyoda, Daisuke Yamada, Yukio Hitsumoto, Eiji Nakata, Toshifumi Ozaki, Takeshi Takarada

Stem cell research & therapy 14 ( 1 ) 34 - 34 2023年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

BACKGROUND: Cell sheet fabrication for articular cartilage regenerative medicine necessitates a large number of chondrocytes of consistent quality as a cell source. Previously, we have developed human-induced pluripotent stem cell (iPSC)-derived expandable PRRX1+ limb-bud mesenchymal cells (ExpLBM) with stable expansion and high chondrogenic capacity, while in this study; our ExpLBM technology was combined with cell sheet engineering to assess its potential as a stable cell source for articular cartilage regeneration. METHODS: ExpLBM cells derived from human-induced pluripotent stem cells (hiPSCs), including 414C2 and Ff-KVs09 (HLA homozygous), were seeded onto a culture plate and two-dimensional chondrogenic induction (2-DCI) was initiated. After 2-DCI, ExpLBM-derived chondrocytes were stripped and transferred to temperature-responsive culture inserts and the chondrocyte sheets were histologically examined or transplanted into osteochondral knee defects of immunodeficient rats. RESULTS: Immunohistochemistry revealed that ExpLBM-derived cell sheets were positive for Safranin O, COL2, and ACAN but that they were negative for COL1 and RUNX2. Furthermore, the engrafted tissues in osteochondral knee defects in immunodeficient rats were stained with SafO, human VIMENTIN, ACAN, and COL2. CONCLUSIONS: The present study is the first to report the chondrocyte sheet fabrication with hiPSC-derived cell source. hiPSC-derived ExpLBM would be a promising cell source for cell sheet technology in articular cartilage regenerative medicine.

DOI： 10.1186/s13287-023-03252-4

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悪性末梢神経鞘腫瘍におけるPRRX1の悪性化因子としての役割

たき平将太, 中田英二, 山田大祐, 片山晴喜, 畑利彰, 藤原智洋, 高尾知佳, 国定俊之, 宝田剛志, 尾崎敏文

日本レックリングハウゼン病学会学術大会プログラム・抄録集 14回 16 - 16 2023年2月

A protocol to induce expandable limb-bud mesenchymal cells from human pluripotent stem cells. 国際誌

Tomoka Takao, Daisuke Yamada, Takeshi Takarada

STAR protocols 3 ( 4 ) 101786 - 101786 2022年12月

記述言語：英語掲載種別：研究論文（学術雑誌）

Here, we present a protocol for the selective differentiation of human pluripotent stem cells mimicking human developmental processes into expandable PRRX1+ limb-bud mesenchymal (ExpLBM) cells. This approach enables expansion through serial passage while maintaining capacity for chondrogenic differentiation. For complete details on the use and execution of this protocol, please refer to Yamada et al. (2021, 2022).

DOI： 10.1016/j.xpro.2022.101786

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Establishment of a human pluripotent stem cell-derived MKX-td Tomato reporter system. 国際誌

Yuki Fujisawa, Lu Ming, Daisuke Yamada, Tomoka Takao, Takeshi Takarada

Stem cell research & therapy 13 ( 1 ) 515 - 515 2022年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Tendon regeneration is difficult because detailed knowledge about tendon progenitor cells (TPCs), which produce tenocytes to repair tendon tissue, has not been revealed. Mohawk homeobox (MKX) is a marker of TPCs or tenocytes, but a human pluripotent stem cell (hPSC)-based reporter system that visualizes MKX+ cells has not been developed. Here, we established an hPSC-derived MKX-tdTomato reporter cell line and tested the induction ratio of MKX-tdTomato+ cells using our stepwise/xeno-free differentiation protocol. MKX-tdTomato+ cells were generated with high efficiency and expressed tendon-specific markers, including MKX, SCX, TNMD, and COL1A1. Our MKX-tdTomato hPSC line would be a useful tool for studying the development or regeneration of tendon tissue.

DOI： 10.1186/s13287-022-03203-5

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Fabrication of scaffold-free mesenchyme tissue bands by cell self-aggregation technique for potential use in tissue regeneration. 国際誌

Tomoyuki Ota, Ryosuke Iwai, Yohei Kitaguchi, Takeshi Takarada, Yoshihiro Kimata

Biomedical materials (Bristol, England) 17 ( 6 ) 2022年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Three-dimensional cell constructs comprising only tissue-specific cells and extracellular matrix secreted by them would be ideal transplants, but their fabrication in a cell aggregation manner without cell scaffolds relies on random cell self-aggregation, making the control of their size and shape difficult. In this study, we propose a method to fabricate band-shaped tissues by inducing the self-aggregation of cell sheets using the developed cell self-aggregation technique (CAT). Acting as cell aggregation stoppers, silicone semicircular pillars were attached to two positions equidistant from both short ends of the rounded rectangular culture groove and coated with a specifically charged biomimetic polymer as a CAT-inducing surface. Mesenchymal stem cells, chondrocytes, and skeletal myoblast cells seeded on the surface of the culture grooves formed band-shaped aggregates between the two aggregation stoppers following spontaneous detachment with aggregation of the cell sheet from the outer edge of the grooves during day one of culture. The aggregated chondrocyte band matured into a cartilage-like plate with an abundant cartilage matrix while retaining its band shape after two weeks of chondrogenic cultivation. Additionally, the aggregates of mesenchymal stem cells and myoblast cell bands could patch the induced collagen membrane derived from rat subcutaneous tissue like a bandage immediately after their formation and successfully mature into fat and muscle tissues, respectively. These results indicate that, depending on the cell type, scaffold-free band-shaped cell aggregates produced by CAT have the potential to achieve tissue regeneration that follows the shape of the defect viain vitromaturation culture orin vivoorganization.

DOI： 10.1088/1748-605X/ac9c7f

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ヒトiPS細胞来肢芽間葉系細胞を用いた硝子軟骨シートの作製

高尾知佳, 佐藤正人, 豊田恵利子, 山田大祐, 中田英二, 尾崎敏文, 宝田剛志

日本整形外科学会雑誌 96 ( 8 ) S1557 - S1557 2022年9月