JP7315923B2 - 臓器オルガノイド及びその製造方法 - Google Patents
臓器オルガノイド及びその製造方法 Download PDFInfo
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Description
<1> 細胞外マトリックス構成タンパク質を含有するゲルの表面上にて、FGFタンパク質の存在下で胚様体を培養する工程を含む、心臓オルガノイド及び/又は肺オルガノイドの製造方法。
<2> FGFタンパク質の存在下で培養する前記工程の後に、更に、FGFタンパク質、BMP及びLIFの存在下で培養する工程を含む、<1>に記載の製造方法。
<3> FGFタンパク質の存在下で培養する前記工程の後に、更に、FGFタンパク質、BMP、LIF及びGSK-3阻害剤の存在下で培養する工程を含む、<1>に記載の製造方法。
<4> 胚様体を培養する前記工程が、前記ゲルの表面上にて、FGFタンパク質及びRho結合キナーゼ阻害剤の存在下で胚様体を培養する工程である、<1>に記載の製造方法。
<5> 前記細胞外マトリックス構成タンパク質が、ラミニン及びエンタクチンからなる群から選択される少なくとも1つを含むタンパク質である、<1>~<4>のうちのいずれか一項に記載の製造方法。
<6> 前記細胞外マトリックス構成タンパク質が、コラーゲン、プロテオグリカンを含まないタンパク質である、<1>~<5>のうちのいずれか一項に記載の製造方法。
<7> 前記胚様体が、LIFの非存在下、多能性幹細胞を浮遊培養してなる細胞塊である、<1>~<6>のうちのいずれか一項に記載の製造方法。
<8> 細胞外マトリックス構成タンパク質を含有する溶液又はゲルと、FGFタンパク質と、胚様体を培養するための培地とを含む、心臓オルガノイド及び/又は肺オルガノイドを製造するためのキット。
<9> BMP、GSK-3阻害剤、LIF及びRho結合キナーゼ阻害剤から成る群より選択される少なくとも一つを更に含む、<8>に記載のキット。
<10> 前記細胞外マトリックス構成タンパク質が、ラミニン及びエンタクチンからなる群から選択される少なくとも1つを含むタンパク質である、<8>又は<9>に記載のキット。
<11> 前記細胞外マトリックス構成タンパク質が、コラーゲン及びプロテオグリカンを含まないタンパク質である、<8>~<10>のうちのいずれか一項に記載のキット。
<12> 多能性幹細胞を浮遊培養により胚様体にするための、LIFを含有しない培地を、更に含む、<8>~<11>のうちのいずれか一項に記載のキット。
<13> 細胞外マトリックス構成タンパク質を含有するゲルの表面上にて、FGFタンパク質の存在下、胚様体を培養してなる、心臓オルガノイド、肺オルガノイド、又はこれらオルガノイドの断片若しくは細胞。
<14> 生体への移植に用いるための、<13>に記載の心臓オルガノイド、肺オルガノイド、又はこれらオルガノイドの断片若しくは細胞。
<15> FGFタンパク質の存在下で培養した後に、更に、FGFタンパク質、BMP及びLIFの存在下で培養してなる、<13>又は<14>に記載の心臓オルガノイド、肺オルガノイド、又はこれらオルガノイドの断片若しくは細胞。
<16> FGFタンパク質の存在下で培養した後に、更に、FGFタンパク質、BMP、LIF及びGSK-3阻害剤の存在下で培養してなる、<13>又は<14>に記載の心臓オルガノイド、肺オルガノイド、又はこれらオルガノイドの断片若しくは細胞。
<17> 前記ゲルの表面上にて、FGFタンパク質及びRho結合キナーゼ阻害剤の存在下で胚様体を培養してなる、<13>又は<14>に記載の心臓オルガノイド、肺オルガノイド、又はこれらオルガノイドの断片若しくは細胞。
<18> 前記細胞外マトリックス構成タンパク質が、ラミニン及びエンタクチンからなる群から選択される少なくとも1つを含むタンパク質である、<13>~<17>のうちのいずれか一項に記載の心臓オルガノイド、肺オルガノイド、又はこれらオルガノイドの断片若しくは細胞。
<19> 前記細胞外マトリックス構成タンパク質が、コラーゲン及びプロテオグリカンを含まない、<13>~<18>のうちのいずれか一項に記載の心臓オルガノイド、肺オルガノイド、又はこれらオルガノイドの断片若しくは細胞。
<20> 前記胚様体が、LIFの非存在下、多能性幹細胞を浮遊培養してなる細胞塊である、<13>~<18>のうちのいずれか一項に記載の心臓オルガノイド、肺オルガノイド、又はこれらオルガノイドの断片若しくは細胞。
<21> 前記多能性幹細胞が、心臓疾患又は肺疾患の罹患者に由来する多能性幹細胞である、<20>に記載の、心臓オルガノイド、肺オルガノイド、又はこれらオルガノイドの断片若しくは細胞。
<22> 化合物の心臓に対する毒性を評価するための方法であって、
<13>~<21>のうちのいずれか一項に記載の心臓オルガノイドと、被験化合物とを接触させ、該心臓オルガノイドの状態を検出する工程と、
前記工程にて検出される状態において、悪化が認められれば、前記被験化合物は心臓に対して毒性を有する化合物であると判定する工程とを、
含む方法。
<23> 化合物の心臓疾患の治療活性を評価するための方法であって、
心臓疾患を呈する<13>~<21>のうちのいずれか一項に記載の心臓オルガノイドと、被験化合物とを接触させ、該心臓オルガノイドの状態を検出する工程と、
前記工程にて検出される状態において、前記心臓疾患に対する治療効果が認められれば前記被験化合物は前記心臓疾患に対する治療活性を有する化合物であると判定する工程とを、
含む方法。
<24> 化合物の肺に対する毒性を評価するための方法であって、
<13>~<21>のうちのいずれか一項に記載の肺オルガノイドと、被験化合物とを接触させ、該肺オルガノイドの状態を検出する工程と、
前記工程にて検出される状態において、悪化が認められれば、前記被験化合物は肺に対して毒性を有する化合物であると判定する工程とを、
含む方法。
<25> 化合物の肺疾患の治療活性を評価するための方法であって、
肺疾患を呈する<13>~<21>のうちのいずれか一項に記載の肺オルガノイドと、被験化合物とを接触させ、該肺オルガノイドの状態を検出する工程と、
前記工程にて検出される状態において、前記肺疾患に対する治療効果が認められれば前記被験化合物は前記肺疾患に対する治療活性を有する化合物であると判定する工程とを、
含む方法。
本発明の心臓オルガノイド及び/又は肺オルガノイド(以下「心臓オルガノイド等」とも総称する)を製造するための方法は、細胞外マトリックス構成タンパク質を含有するゲルの表面上にて、FGFタンパク質の存在下、胚様体を培養することを特徴とする。
また、本発明は、上述の心臓オルガノイド等の製造に用いられるキットを提供する。本発明のキットは、少なくとも、上述の細胞外マトリックス構成タンパク質を含有するゲルと、胚様体を培養するための上述の培地(心臓オルガノイド等への分化誘導用培地)と、当該培地に添加するFGFタンパク質とを含むものである。また、本発明のキットは、前記ゲルの代わりに又はそれと併せて、ゲル化する前の、細胞外マトリックス構成タンパク質を含有する溶液を含むものであってもよい。
後述の実施例において示すとおり、上述の方法によって製造される心臓オルガノイド及び肺オルガオルガノイドは、生体内におけるそれらの機能を担う構造を再現している。特に、心臓オルガノイドは、各種細胞の部域性は生体内におけるそれらを忠実に再現しており、さらに心筋収縮等の機能も発揮するものである。そして、かかる臓器オルガノイドを用いることにより、後述のとおり、対応する臓器に関する疾患を治療、改善又は予防するための化合物又は該臓器に対して毒性を有する化合物のスクリーニング、更には前記臓器に関する疾患等の再生医療を行なうことができる。
後述の実施例において示すとおり、本発明の心臓オルガノイドは、生体内における心臓の構造及び機能を忠実に再現している。また、本発明の肺オルガノイドは、その機能を発揮する上で重要な構造である肺胞を備える。そのため、これら臓器オルガノイドは、化合物の対応する臓器に対する毒性活性を評価する上で有用である。特に、心臓に関し、薬物によるQT延長は突然死にも関係しているので有用であると思われる。また、肺オルガノイドに関しては、新たに開発された薬剤(特に、抗がん剤)が、薬剤性肺炎又は肺線維症の副作用を持つのかについての評価(肺毒性の評価)にも、有効である。
トリプシン処理した野生型マウス由来のES細胞(由来の異なる3細胞株)及びαMHC-GFP ES細胞を、0.2%ゼラチンコートウェルに播種し、37℃にて45分間インキュベーションした。次いで、浮遊したES細胞を回収し、1000rpmの遠心処理に5分間供した。
心臓オルガノイドは、培養10~15日後に回収し、プラスチック組織モルド(クリオディッシュ、株式会社硝英製作所製)中に、ティシュー・テック(登録商標)OCTコンパウンド包埋し、凍結した。凍結切片は、-16℃のクリオスタットにて厚さが5~7μmになるよう調製し、MASコートガラススライド(松浪硝子工業株式会社製)又はポリLリジンコートスライド(シグマアルドリッチ社製)に移した。心臓オルガノイドへの分化誘導に供しなかった胚様体も回収し、上記同様の方法にて、OCTに凍結包埋し、切片に調製した。
Tbx5(アブカム社製、ab137833)、心筋トロポニン I(アブカム社製、ab47003)、Nkx2-5(アブカム社製、ab91196)、ネスチン(アブカム社製、ab105389)、Oct3/4(Santa Cruz Biotech社製),PECAM(BD社製)、Mlc2a(Synaptic System社製、#311 011)、Mlc2v(Synaptic System社製、#310 003)、SM-MHC(アブカム社製、R&D Systems社製)及びαSMA(ab5694)。
心臓オルガノイドは、PBSにて2回洗浄し、1.8mM Ca2+含有タイロード溶液にて新たに希釈した、4μM 緑色蛍光の細胞内カルシウム指示薬 Fluo8 AM(アセトキシメチル)又はFluo8 AM/F127に、37℃、15~30分間浸した。その後、PBSにて2回洗浄した。そして、心臓オルガノイドを、200μL タイロード溶液にて処理し、マウントした後、蛍光顕微鏡(Keyence社製)にて観察した。
<心臓オルガノイドの形成>
図1の上段において示すとおり、胚発生において、心臓の形成は、血管内皮細胞、心筋細胞及び平滑筋細胞といった多種多様な細胞への分化を伴う特色のある形態変化を示す。先ず、胎生7.5日目において、1次心臓領域及び2次心臓領域を含む心原基の形成は、ブラキウリの発現によって誘導される、心臓中胚葉遺伝子(Mesp1、Flk1、Pdgfra)の発現によって特徴づけられる。次いで、胎生8日目には、線状の拍動する心筒を形成するに至る。さらに、この内層に血管内皮細胞を、外層に心筋細胞を有する管状形態をとる心筒は、胎生9.5日目に、拍動力又は流出路(OFT)によって、ルーピングする。そして、4つの心腔(右心房、左心房、右心室及び左心室)が、胎生10.5日迄に形成され、胎生14.5日迄に成熟されることになる。
<心臓オルガノイドにおける心臓細胞特異的マーカーの検出>
次に、心臓オルガノイドにおいて、胎児の心臓形成において必要である、心臓特異的転写因子及び心臓構造遺伝子が、これらオルガノイドにおいて適切に発現しているかどうかを決定するため、形態学的に分析した(n=25)。
<心臓オルガノイドにおける機能的な収縮の検出>
自発性の拍動が心臓オルガノイドにおいて検出された時、カルシウム振動といった特定の心臓機能が得られているかどうかについて評価した。すなわち、細胞内カルシウム指示薬を用い、Ca2+レベルを測定することによって、解離が生じていない完全な3D心臓オルガノイドを分析した(n=10)。
<透過型電子顕微鏡による、心臓オルガノイドの解析>
透過型電子顕微鏡(TEM)解析のために、胎生10.5日目、11.5日目のマウス心臓と、心臓オルガノイドから、90nm超薄切片を各々調製した。次いで、酢酸ウラニル及びクエン酸鉛にて二重染色し、観察及び撮影を行った。得られた結果を図28に示す。
<フローサイトメトリー解析による心筋細胞の定量化>
フローサイトメトリー解析のため、胚様体、並びに培養後8日目及び12日目の心臓オルガノイドを、各々コラゲナーゼ(1mg/ml)にて37℃15分間処理し、次いで、TrpLE(Gibco社製)にて37℃10~15分間処理した。そして、当該胚様体及び心臓オルガノイド、並びに生体の心臓を、1細胞ずつに分離するよう、各々ピペッティング処理に供した。このようにして調製した細胞を4%PFA/PBSにて、4℃10分間処理し、固定化した。次いで、これら細胞を、4%FBS/PBS/0.5%サポニンにて希釈した抗体(Alexa647結合抗cTnT抗体)にて、4℃30分間処理し、染色した。そして、FACS Aria II〈BD社製)を用いた解析に供した。その結果、図29に示すとおり、cTnT(心筋トロポニン)陽性の心筋細胞における有意な増加が、心臓オルガノイドにおいて認められた。
<RNA-seqデータを用いた主成分分析(PCA)>
胚様体、並びに培養後9日目及び13日目の心臓オルガノイドを回収し、液体窒素にて新鮮なまま凍結保存した。また、各発達段階にて解剖し、得られた胎児の心臓も回収し、前記胚様体等と同様に凍結した。そして、これら各凍結サンプルから、AllPrep DNA/RNAマイクロキット(QIAGEN社製)を用い、そのプロトコールに沿ってトータルRNAを抽出した。次いで、RNA-seq解析のためのライブラリーを、KAPA スタンダードmRNA-Seq キット(KAPA Biosystems社製)を用いて調製した。得られたライブラリーは、HiSeq 1500(single end 50bp reads with HiSeq SR Rapid Cluster Kit v2 and HiSeq Rapid SBS Kit v2,Illumina社製)によるシークエンシングに供した。得られたシークエンスデータに関し、Bowtie2を用い、マウスレファレンスゲノムシークエンス(GRCm38/mm10)に対するマッピングを行なった。そして、Bioconductor package DEGseqを用い、各遺伝子におけるread数及びRPKM値を求めた。
<心臓オルガノイドにおける伝導系についての確認>
心臓オルガノイドにおいて、心筋トロポニンTとPurkinje fiberで発現するTRPM4の発現を、蛍光免疫染色によって検出した。なお、蛍光免疫染色は、一次抗体として、cTnT(ab8295)及びTRPM4(ABN418)を用いた以外は、上記(蛍光免疫染色)と同様の方法にて行なった。
<心臓オルガノイドにおける成熟細胞についての確認>
心臓オルガノイドにおいて、カリウムチャネル iK1の発現を、蛍光免疫染色によって検出した。なお、カリウムチャネル iK1は、活動電位の立ち上がり速度や伝導速度を早くするために必要であり、成熟な心筋細胞のみで検出される。また、蛍光免疫染色は、一次抗体として、KCNN4(iK1,GTX54786)を用いた以外は、上記(蛍光免疫染色)と同様の方法にて行なった。
<心臓オルガノイドにおけるT管についての確認>
心毒性検査に用いられる心筋細胞の新しい評価基準(FDA)として、T管(Transverse tubule)が重要視されている。しかしながら、ヒトiPS由来心筋細胞においては観察されておらず、それが問題となっている(Yang,X.ら、Circ Res 114(3)511-23(2014) 参照)。
<心臓オルガノイドの電気生理学的評価>
心臓オルガノイドを電気生理学的に評価すべく、光学マッピング(Optical mapping)を、以下に示す方法にて行なった。なお、電気生理的評価法として他に、多点平面電極(MEA)を用いた細胞外電位記録法があるが、この方法では不整脈の予測が出来ない。それに対し、光学マッピングを用いると、自発興奮及び膜電位だけではなく、波形の間隔が不規則的に変化することを示すことができるため、不整脈の予測も可能である。
<心臓オルガノイドの薬剤応答性の評価>
上記にて示したとおり、本発明の心臓オルガノイドは、成熟度が高い。そのため、薬剤安全性評価のための心毒性検査等に有効な材料になり得る。そこで、その有効性を確認すべく、心臓機能異常を引き起こす薬剤を用い、心臓オルガノイドにおける薬剤の応答性を評価した。
<心臓オルガノイド形成における、細胞外マトリックス構成タンパク質についての検討>
上記(細胞培養)に記載の方法と同様にして、マウスES細胞を、白血病阻害因子(LIF)の非存在下、低結合性U底96ウェルプレートにて培養し、完全な胚様体を得た。そして、得られた胚様体を、結合組織における細胞外マトリックス(ECM)の成分を含む、ラミニン411((Nippi社、iMatrix-411;ヒトラミニン411タンパク質のインテグリン結合部位(E8切片)の精製品)、ラミニン221(Nippi社、iMatrix-221;ヒトラミニン221タンパク質のインテグリン結合部位(E8切片)の精製品)、又はラミニン221及び411を同量混ぜた混合物の表面上にて、外因性Fgfシグナル(FGF4)の存在下、培養した。ラミニンのコーティングは、それぞれ10.7μg/cm2にて行なった。胚様体の培養期間と培地交換も、上記(細胞培養)の記載に沿って行ない、当該培養後、蛍光免疫染色等の更なる解析のため、心臓オルガノイドを回収した。
<ヒトiPS細胞を用いた、心臓オルガノイド及び肺オルガノイドの形成>
上述のマウスES細胞の代わりに、ヒトiPS細胞を用いても、心臓オルガノイドを形成できることを、以下に示す方法にて確認した。
Claims (6)
- 細胞外マトリックス構成タンパク質を含有するゲルの表面上にて、FGFタンパク質の存在下で、多能性幹細胞由来の胚様体を培養する工程を含む、臓器オルガノイドの製造方法であって、
(a)前記細胞外マトリックス構成タンパク質はラミニン111又はラミニン411であり、前記工程の後に、更に、FGFタンパク質、GSK-3阻害剤、BMP4及びLIFの存在下で培養する工程を含み、前記臓器オルガノイドが心臓オルガノイドである方法、又は、
(b)前記細胞外マトリックス構成タンパク質がラミニン111及びエンタクチンであり、FGFタンパク質がFGF4タンパク質であり、前記多能性幹細胞がヒトiPS細胞であり、前記臓器オルガノイドが肺オルガノイドである方法。 - (b)において、胚様体を培養する前記工程が、前記ゲルの表面上にて、前記FGFタンパク質に加えてRho結合キナーゼ阻害剤の存在下で、前記胚様体を培養する工程である、請求項1に記載の製造方法。
- 前記胚様体が、LIFの非存在下、前記多能性幹細胞を浮遊培養してなる細胞塊である、請求項1又は2に記載の製造方法。
- 前記細胞外マトリックス構成タンパク質を含有する溶液又は前記ゲルと、前記FGFタンパク質と、前記胚様体を培養するための培地とを含む、請求項1~3のうちのいずれか1項に記載の方法にて、臓器オルガノイドを製造するためのキット。
- BMP4、GSK-3阻害剤、LIF及びRho結合キナーゼ阻害剤からなる群より選択される少なくとも一つを更に含む、請求項4に記載のキット。
- 前記多能性幹細胞を浮遊培養により胚様体にするための、LIFを含有しない培地を、更に含む、請求項4又は5に記載のキット。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005017131A3 (en) | 2003-08-14 | 2005-11-24 | Gouvernment Of The United Stat | Methods for the differentiation of human stem cells |
US20170002330A1 (en) | 2015-05-11 | 2017-01-05 | The Trustees Of Columbia University In The City Of New York | Engineered adult-like human heart tissue |
US20170275592A1 (en) | 2014-11-27 | 2017-09-28 | Koninklijke Nederlandse Akademie Van Wetenschappen | Culture medium |
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AU2016226178B2 (en) * | 2015-03-03 | 2021-07-29 | President And Fellows Of Harvard College | Methods of generating functional human tissue |
JP6998772B2 (ja) * | 2015-04-09 | 2022-02-04 | ビオラミナ アーベー | 神経変性疾患の処置における使用のための幹細胞由来のドパミン作用性細胞を生成するための方法および組成物 |
-
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005017131A3 (en) | 2003-08-14 | 2005-11-24 | Gouvernment Of The United Stat | Methods for the differentiation of human stem cells |
US20170275592A1 (en) | 2014-11-27 | 2017-09-28 | Koninklijke Nederlandse Akademie Van Wetenschappen | Culture medium |
US20170002330A1 (en) | 2015-05-11 | 2017-01-05 | The Trustees Of Columbia University In The City Of New York | Engineered adult-like human heart tissue |
Non-Patent Citations (4)
Title |
---|
Biomaterials,2017年07月12日,Vol. 142,pp. 112-123 |
eLIFE,2015年,Vol. 4,e05098, pp. 1-25 |
PLOS ONE,2014年,Vol. 9,e94764, pp. 1-10 |
The Journal of Clinical Investigation,2005年,Vol. 115,pp. 1724-1733 |
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