研究者詳細 - 寳田　剛志

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

PubMed

researchmap

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

PubMed

researchmap

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

PubMed

researchmap

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

PubMed

researchmap

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

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, 山田大祐, 高尾知佳, 中田英二, 近藤宏也, 佐藤浩平, 畑利彰, 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 96 ( 8 ) S1768 - S1768 2022年9月

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

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, 山田大祐, 高尾知佳, 中田英二, 近藤宏也, 佐藤浩平, 畑利彰, 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 96 ( 8 ) S1768 - S1768 2022年9月

Vestigial-Like 3 Plays an Important Role in Osteoblast Differentiation by Regulating the Expression of Osteogenic Transcription Factors and BMP Signaling. 国際誌

Haoze Yuan, Mika Ikegame, Yoko Fukuhara, Fumiko Takemoto, Yaqiong Yu, Jumpei Teramachi, Yao Weng, Jiajie Guo, Daisuke Yamada, Takeshi Takarada, Ying Li, Hirohiko Okamura, Bin Zhang

Calcified tissue international 111 ( 3 ) 331 - 344 2022年6月

記述言語：英語掲載種別：研究論文（学術雑誌）

Our previous gene profiling analysis showed that the transcription cofactor vestigial-like 3 (VGLL3) gene expression was upregulated by mechanical tension in the mouse cranial suture, coinciding with accelerated osteoblast differentiation. Therefore, we hypothesized that VGLL3 plays a significant role in osteogenic differentiation. To clarify the function of VGLL3 in osteoblasts, we examined its expression characteristics in mouse bone tissue and the osteoblastic cell line MC3T3-E1. We further examined the effects of Vgll3 knockdown on osteoblast differentiation and bone morphogenetic protein (BMP) signaling. In the mouse cranial suture, where membranous ossification occurs, VGLL3 was immunohistochemically detected mostly in the nucleus of osteoblasts, preosteoblasts, and fibroblastic cells. VGLL3 expression in MC3T3-E1 cells was transient and peaked at a relatively early stage of differentiation. RNA sequencing revealed that downregulated genes in Vgll3-knockdown cells were enriched in gene ontology terms associated with osteoblast differentiation. Interestingly, most of the upregulated genes were related to cell division. Targeted Vgll3 knockdown markedly suppressed the expression of major osteogenic transcription factors (Runx2, Sp7/osterix, and Dlx5) and osteoblast differentiation. It also attenuated BMP signaling; moreover, exogenous BMP2 partially restore osteogenic transcription factors' expression in Vgll3-knockdown cells. Furthermore, overexpression of Vgll3 increased the expression of osteogenic transcription factors. These results suggest that VGLL3 plays a critical role in promoting osteoblast differentiation and that part of the process is mediated by BMP signaling. Further elucidation of VGLL3 function will increase our understanding of osteogenesis and skeletal disease etiology.

DOI： 10.1007/s00223-022-00997-7

PubMed

researchmap

Identification of Surface Antigens That Define Human Pluripotent Stem Cell-Derived PRRX1+Limb-Bud-like Mesenchymal Cells. 国際誌

Daisuke Yamada, Tomoka Takao, Masahiro Nakamura, Toki Kitano, Eiji Nakata, Takeshi Takarada

International journal of molecular sciences 23 ( 5 ) 2022年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Stem cell-based therapies and experimental methods rely on efficient induction of human pluripotent stem cells (hPSCs). During limb development, the lateral plate mesoderm (LPM) produces limb-bud mesenchymal (LBM) cells that differentiate into osteochondroprogenitor cells and form cartilage tissues in the appendicular skeleton. Previously, we generated PRRX1-tdTomato reporter hPSCs to establish the protocol for inducing the hPSC-derived PRRX1+ LBM-like cells. However, surface antigens that assess the induction efficiency of hPSC-derived PRRX1+ LBM-like cells from LPM have not been identified. Here, we used PRRX1-tdTomato reporter hPSCs and found that high pluripotent cell density suppressed the expression of PRRX1 mRNA and tdTomato after LBM-like induction. RNA sequencing and flow cytometry suggested that PRRX1-tdTomato+ LBM-like cells are defined as CD44high CD140Bhigh CD49f-. Importantly, other hPSC lines, including four human induced pluripotent stem cell lines (414C2, 1383D2, HPS1042, HPS1043) and two human embryonic stem cell lines (SEES4, SEES7), showed the same results. Thus, an appropriate cell density of hPSCs before differentiation is a prerequisite for inducing the CD44high CD140Bhigh CD49f- PRRX1+ LBM-like cells.

DOI： 10.3390/ijms23052661

PubMed

researchmap

Mouse Model for Optogenetic Genome Engineering.

Tomoka Takao, Daisuke Yamada, Takeshi Takarada

Acta medica Okayama 76 ( 1 ) 1 - 5 2022年2月

記述言語：英語掲載種別：研究論文（学術雑誌）

Optogenetics, a technology to manipulate biological phenomena thorough light, has attracted much attention in neuroscience. Recently, the Magnet System, a photo-inducible protein dimerization system which can control the intracellular behavior of various biomolecules with high accuracy using light was developed. Furthermore, photoactivation systems for controlling biological phenomena are being developed by combining this technique with genome-editing technology (CRISPR/Cas9 System) or DNA recombination technology (Cre-loxP system). Herein, we review the history of optogenetics and the latest Magnet System technology and introduce our recently developed photoactivatable Cre knock-in mice with temporal-, spatial-, and cell-specific accuracy.

DOI： 10.18926/AMO/63202

PubMed

researchmap

骨系統疾患特異的iPSC由来ヒト肢芽間葉系細胞を用いたハイスループット薬剤スクリーニングシステムの構築

山田大祐, 髙尾知佳, 中村正裕, Ming Lu, 戸口田淳也, 宝田剛志

日本薬理学会年会要旨集 95 2-O-087 2022年

記述言語：日本語出版者・発行元：公益社団法人日本薬理学会

[Background] To achieve the disease modeling of skeletal dysplasia, human induced pluripotent stem cells (iPSCs) will be useful but the induction method for limb bud mesenchymal (LBM) cells, which can give rise to most of the future limb elements - bone, cartilage and tendon/ligament, has not been established. In this study, we developed the induction and expansion protocol of LBM cells from human iPSCs, and a high-throughput drug screening system using human LBM cells differentiated from iPSC which is derived from patients with skeletal dysplasia.

[Method] Paired-related homeobox 1 (PRRX1) serves as a marker for the LBM. To assess the induction efficiency of PRRX1 positive cells during each differentiation step, we established PRRX1-tdTomato reporter human iPSC line. Lateral plate mesoderm cells (LPM) derived from PRRX1-tdTomato reporter were used to establish the LBM-inducing method that recapitulates human developmental process. The chondrogenic capacity of expandable LBM (ExpLBM) cells was assessed using our two- or three- dimensional chondrogenic induction method (2DCI or 3DCI). In addition, ExpLBM cells differentiated from iPSCs derived from patients with type II collagenopathy (COL2pathy), one of the skeletal dysplasia arising from mutations in COL2A1, were used to develop high-throughput screening system.

[Results] By activating WNT signaling and inhibiting BMP/TGFb/headge hog signaling pathways, almost all LPM cells could be induced to PRRX1 positive LBM cells. Interestingly, we found the defined culture method that can not only stably expand LBM cells (ExpLBM) but also maintain their PRRX1 expression. ExpLBM formed Alcian Blue positive nodules under 2DCI condition and Safranin O positive cartilaginous particles under 3DCI condition. 2DCI-based high-throughput screening system found that several chemicals improved the chondrogenic capacity of ExpLBM derived from COL2pathy patient.

[Conclusion] ExpLBM cells will be a potential tool to study human bone development, cartilage regeneration and drug discovery using disease-specific human iPSCs.

DOI： 10.1254/jpssuppl.95.0_2-o-087

researchmap

O‐GlcNAcylation drives calcium signaling toward osteoblast differentiation: A bioinformatics‐oriented study

Yao Weng, Ziyi Wang, Yoko Fukuhara, Airi Tanai, Mika Ikegame, Daisuke Yamada, Takeshi Takarada, Takashi Izawa, Satoru Hayano, Kaya Yoshida, Hiroshi Kamioka, Hirohiko Okamura

BioFactors 47 ( 6 ) 992 - 1015 2021年8月

　前のページ - 次のページ　

<寳田 剛志>

論文 - 寳田 剛志

<寳田　剛志>

論文 - 寳田　剛志