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Specific induction and long-term maintenance of high purity ventricular cardiomyocytes from human induced pluripotent stem cells

Fukushima, Hiroyuki 京都大学 DOI:10.14989/doctor.r13443

2021.09.24

概要

【背景・目的】ヒト iPS 細胞からの心筋細胞は、心臓毒性スクリーニング、創薬、疾患モデリングなど、さまざまな分野への応用が期待されており、高純度のヒト iPS 細胞由来心筋細胞の安定した作製は、ますます需要が高まっている。しかしながら、現在のヒト iPS 細胞からの心筋細胞の分化誘導方法では、心筋細胞と非心筋細胞が混在しながら作製されるため、作製の最終段階に非心筋細胞を除く精製過程が必要となっている。また一旦精製しても次第に純度が低下する。作製された心筋細胞においては、生体の心室筋細胞様、心房筋細胞様、ペースメーカー細胞様など、様々な性質を有する細胞が混ざり合って構成されている。
本研究では、従来の心筋分化誘導方法を手掛かりに、非心筋細胞を除く精製過程なしに高純度の心室筋細胞を作製する方法を開発することを試みた。

【方法・結果】まず、これまでの心血管系の分化誘導方法の知見をもとに、ヒト iPS 細胞を無血清二次元培養下において、成長因子であるActivin A, BMP4, bFGF を用いて、心筋細胞への分化能を有する中胚葉細胞集団（血小板由来成長因子受容体-α（PDGFRα）陽性細胞）を誘導および収集した。収集したPDGFRα 陽性細胞は、2 種類のWnt シグナル阻害剤 (XAV 及びIWP4)を用いて強力に心筋細胞へ誘導することにより、約95％の細胞が心筋細胞特異的マーカーであるCardiac troponin T 陽性の細胞に分化した（無処理; 7.1 ± 3.1%, XAV 単独; 74.5 ± 6.5%, IWP4 単独; 72.2 ± 6.8%, XAV+IWP4 併用; 94.1 ± 2.0%)。この心筋分化能を有する前駆段階の細胞の収集と強力な心筋への分化誘導刺激によって、非心筋細胞を除去する過程なしに高純度の心筋細胞が安定して得られた。さらに 200 日を超える長期間においても高純度を維持し培養し続けることが可能であった。
さらに、作製した心筋細胞の性質を詳細に解析した。得られた心筋細胞は、ほぼ全ての細胞において、心室筋の特異的マーカーであるミオシン軽鎖2v を発現し、長期間の培養によって、その発現はより顕著になることを認めた。また、長期培養した心筋細胞は、横紋（サルコメア）構造の成熟化（Z 帯、M 帯などの形成、サルコメア長の延長(培養初期: 1.37 ± 0.10 μm, 200 日以上培養: 1.91 ± 0.15 μm)）、ミトコンドリアの増加・成熟化、さらには成体の成熟化した心筋細胞の構造であるT 管様構造を認めた。
電気生理学的解析においても、得られた心筋細胞の90％以上は、心室筋様の活動電位(AP)波形を示した。また長期間の培養によって、心室筋細胞の成熟の特徴である最大拡張期電位の低下、電位ピーク値、AP 増幅量、及び AP 上昇の最高速度 (dV/dtmax)の増加を示した。さらに成熟した心室筋細胞が有する IKs 電流を阻害し QT 延長を示すクロマノールに対しても、生体の心室筋と同様の反応を示した。

【結語】ヒト iPS 細胞から非心筋細胞の除去過程なしに高純度の心室筋細胞を安定して作製する方法を開発した。本方法では、高純度状態で 200 日以上の長期間維持培養が可能であり、長期間の培養により形態学的および電気生理学的成熟が確認できた。この作製技術は、心臓毒性試験、疾患モデリングなどの新たな研究用ツールとしての応用が期待される。

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