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大学・研究所にある論文を検索できる 「In vivo regeneration of rat laryngeal cartilage with mesenchymal stem cells derived from human induced pluripotent stem cells via neural crest cells」の論文概要。リケラボ論文検索は、全国の大学リポジトリにある学位論文・教授論文を一括検索できる論文検索サービスです。
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In vivo regeneration of rat laryngeal cartilage with mesenchymal stem cells derived from human induced pluripotent stem cells via neural crest cells

Yoshimatsu, Masayoshi 京都大学 DOI:10.14989/doctor.k23417

2021.07.26

概要

【背景】
　喉頭・気管は軟骨組織(硝子軟骨)により枠組みを保持されており、呼吸、発声、嚥下機能を担う重要な臓器である。しかし、外傷や、炎症性疾患・悪性腫瘍に対する手術などで軟骨が欠損した場合、枠組みが維持できなくなり、その機能は大きく損なわれる。硝子軟骨は再生能力に乏しいため、喉頭・気管の軟骨欠損に対してこれまで様々な再生医療研究が行われてきたが、治療法の確立には至っていない。近年、細胞治療のための細胞源として、軟骨細胞への分化能を示し，増殖能を持つ間葉系幹細胞 (mesenchymal stem cell: MSC)が着目されており、自家MSC を用いた軟骨組織再生効果が示されている。しかし、自家 MSC などの初代培養 MSC の場合、増殖能や分化能が継代とともに失われるため移植に必要な十分量の細胞を得ることが難しい場合がある。そこで無限増殖能、多分化能を有するinduced pluripotent stem cell (iPS)細胞に着目した。
　甲状軟骨は神経堤細胞 (neural crest cell: NCC)由来MSC を起源とすると考えられている。この発生過程を模倣し作製された、NCC を介したヒトiPS 由来MSC (iMSC)のin vitro での軟骨分化能はすでに示されているが、in vivo における軟骨分化能や軟骨組織再生能は未だ示されていない。そこで本研究では NCC を介して誘導したiMSC の軟骨再生効果を、ラット甲状軟骨欠損モデルを用いて検討した。

【方法】
　ヒトiPS 細胞からNCC を介してiMSC への分化誘導を行った。将来の臨床応用の可能性を考えて、動物由来成分不含、無血清の培養条件を選択し、誘導された細胞はフローサイトメトリー解析により表面抗原の発現を確認することにより iMSC への分化を確認した。
　iMSC は温度感受性ウェルプレートで培養した後、低接着性ウェルプレート上でさらに培養を行い、細胞集塊を形成させた。免疫不全ラットの甲状軟骨両翼に直径1 mm の円盤状軟骨欠損部位を作製した後、左側にのみiMSC の細胞集塊を3 個移植し、4 週、8 週後に組織学的評価を行った。

【結果】
　移植した14 匹全例で移植部位に一致してヒト核抗体 (human nuclear antigen: HNA)陽性細胞を認め、移植細胞の生着が確認された。右側には欠損部位に軟骨組織を認めなかったが、左側では術後4 週で5/7 例、術後8 週で5/7 例においてHE 染色で正常軟骨に特徴的な小腔形成を示す軟骨様組織を甲状軟骨の切除断端に連続して認めた。また、軟骨様組織再生部位に一致してHNAおよび軟骨系細胞マーカーであるSOX9抗体の共発現を認め、軟骨の細胞外基質の評価に用いられるAlcian Blue 染色、Safranin O 染色では、同部位に正常硝子軟骨と類似した染色陽性組織を認めた。さらに、I 型・II 型コラーゲン抗体を用いた免疫組織化学ではHNA陽性細胞の周囲にもII型コラーゲン抗体陽性、I 型コラーゲン抗体陰性の硝子軟骨組織再生を認めた。

【考察】
　本実験結果から、移植したiMSC の一部が軟骨系細胞へ組織内で分化し、それらがラット甲状軟骨の再生を促進したことが示唆された。ホスト軟骨由来の細胞外基質がMSCの軟骨化を促進することが他臓器では報告されており、本研究ではすべての軟骨様再生組織がラット甲状軟骨の切除断端に連続するように認められたことから、ホスト甲状軟骨由来成分が iMSC の軟骨化に寄与したことが推察された。また、MSC 由来の細胞外基質も軟骨分化や軟骨組織再生を促すともいわれている。本研究で用いた細胞集塊作製方法はMSC による細胞外基質産生を促すことが報告されており、本研究ではiMSC の細胞集塊由来の細胞外基質もiMSC の軟骨分化に寄与した可能性がある。

【結論】
　ヒトiPS 細胞からNCC を介して誘導したiMSC のラット甲状軟骨再生効果が示された。本研究は気管喉頭軟骨欠損に対する新たな喉頭軟骨再生方法の可能性を示した。

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