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Disease modeling of pulmonary fibrosis using human pluripotent stem cell-derived alveolar organoids

Suezawa, Takahiro 京都大学 DOI:10.14989/doctor.r13502

2022.09.26

概要

肺線維症は、労作時呼吸困難や乾性咳嗽を主症状とする慢性進行性の間質性肺疾患である。肺線維症の発症には、呼吸機能を担う肺胞上皮細胞が繰り返し傷害を受けることによって、傷害修復を担う線維芽細胞が異常に活性化し、組織収縮や細胞外基質の過剰沈着をきたすことが関与している。肺線維症の疾患モデルでは肺胞上皮細胞と線維芽細胞の相互作用（上皮―間葉相互作用）が重要と考えられている一方で、この相互作用を培養皿の中で再現することは困難だったため、肺胞上皮細胞の傷害や上皮―間葉相互作用を標的とする肺線維症の治療薬開発には限界があった。近年、ヒトiPS細胞由来の肺胞上皮細胞と肺線維芽細胞を肺胞オルガノイドとして共培養することにより、組織幹細胞であるII型肺胞上皮細胞と生体内でガス交換を担うI型肺胞上皮細胞を共存して培養できることが報告されている。本研究では、この培養法を肺線維症の疾患モデリングに応用することを考案した。

まず、肺胞オルガノイドに線維化誘導剤であるブレオマイシンを添加した結果、溶媒のみを添加した対照群に比べて、線維芽細胞の活性化を伴う3次元培養ゲルの顕著な収縮を見出した。この収縮反応は線維芽細胞単独の3次元培養ゲルではほとんど観察されなかったことから、上皮―間葉相互作用を介した線維芽細胞の活性化が関与している可能性が示唆された。次に、ブレオマイシン処理した肺胞オルガノイド中の肺胞上皮細胞の表現型を解析したところ、II型肺胞上皮細胞の細胞老化や、炎症性サイトカインの産生、I型肺胞上皮細胞への分化異常といった肺線維症患者の肺胞上皮細胞と類似した表現型が観察された。これらを指標として化合物スクリーニングを試みたところ、TGF-βのI型受容体であるALK5の阻害剤を添加することにより、病態表現型を改善できることを見出した。また、肺胞オルガノイド内のII型肺胞上皮細胞においてTGFB1の発現が上昇していること、TGF-βの活性化因子であるintegrinαVβ6が上皮細胞特異的に発現していること、integrinαVβ6の拮抗薬も上記の病態表現型に対して同様の改善効果を示すことから、上皮細胞に依存したTGF-βシグナルの活性化が起こっていることが示された。最後に、ブレオマイシンによる肺胞オルガノイド中の線維芽細胞の活性化が、TGF-βシグナルの阻害によって抑制されるかどうかを評価した。ALK5阻害剤は、ブレオマイシンによる肺胞オルガノイド培養ゲルの収縮反応を抑制し、ブレオマイシン添加によって上昇したACTA2,CNN1,TAGLN,MYH11等の線維芽細胞の収縮関連の遺伝子発現を低下させた。また、プロテオミクス解析においてALK5阻害剤は、ブレオマイシンによって蓄積した細胞外基質やコラーゲンなどの合成酵素の発現量を低下させることも見出した。

以上により、iPS細胞由来の肺胞オルガノイドを活用することで、培養皿の中で肺線維症の病態を再現できること、また治療薬の候補化合物を評価できることが示された。この疾患モデルによりヒトにおける肺胞上皮細胞の傷害や、上皮―間葉相互作用を標的とした新規の肺線維症治療薬の開発が可能になることが期待される。

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