JP2019534006A - 肝臓オルガノイド疾患モデルおよびその作製方法 - Google Patents
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Abstract
Description
本出願は、2016年11月4日に出願された米国仮特許出願第62/471,371号、および2016年6月9日に出願された第62/517,414号の優先権および利益を主張するものであり、これらの各内容は全ての目的のために参照によりその全てが本明細書に組み込まれる。
いくつかの実施形態では、1つのステップは、多能性であるかまたは多能性になるように誘導され得る幹細胞を得ることである。いくつかの実施形態では、多能性幹細胞は胚性幹細胞に由来し、また、この胚性幹細胞は哺乳動物初期胚の全能性細胞に由来し、インビトロで無限の未分化増殖が可能である。胚性幹細胞は、初期段階の胚である胚盤胞の内部細胞塊に由来する多能性幹細胞である。胚盤胞から胚性幹細胞を誘導するための方法は当技術分野において周知である。ヒト胚性幹細胞H9(H9−hESC)は、本明細書に記載されている例示的な実施形態において使用されるが、本明細書に記載されている方法およびシステムは任意の幹細胞に適用可能であることは当業者には理解されよう。
いくつかの実施形態では、iPSCは、成体線維芽細胞などの非多能性細胞への特定の幹細胞関連遺伝子のトランスフェクションによって誘導される。トランスフェクションは典型的には、レトロウイルスのようなウイルスベクターを通して達成される。トランスフェクトされた遺伝子はマスター転写調節因子Oct−3/4(Pouf51)およびSox2を含むが、他の遺伝子が誘導の効率を高めることが示唆されている。3〜4週間後、少数のトランスフェクトされた細胞が多能性幹細胞と形態学的および生化学的に類似するようになり、通常は形態学的選択、倍加時間、またはレポーター遺伝子および抗生物質選択を通じて単離される。本明細書中で使用されるとき、iPSCには、第一世代iPSC、マウスにおける第二世代iPSC、およびヒト人工多能性幹細胞が含まれるが、これらに限定されない。いくつかの実施形態では、レトロウイルス系を用いて、4つの中心遺伝子:Oct3/4、Sox2、Klf4、およびc−Mycを用いて、ヒト線維芽細胞を多能性幹細胞に形質転換する。別の実施形態では、レンチウイルス系を用いて体細胞をOCT4、SOX2、NANOG、およびLIN28で形質転換する。発現がiPSCにおいて誘導される遺伝子には、Oct−3/4(例えば、Pou5fl);Sox遺伝子ファミリーの特定のメンバー(例えば、Sox1、Sox2、Sox3、およびSox15);Klfファミリーの特定のメンバー(例えば、Klf1、Klf2、Klf4、およびKlf5)、Mycファミリーの特定のメンバー(例えば、C−myc、L−myc、およびN−myc)、Nanog、およびLIN28が含まれるが、これらに限定されない。
ヒトiPSCからのRAに基づく肝臓オルガノイドモデルの生成
RAシグナル伝達が間質細胞に対する系統分化の決定に影響を与えるかどうかを明らかにするために、出願人は、RAの一過性誘導によるヒトiPSCからの肝臓オルガノイドモデルを確立した。以前に記載されているように(Spence et al.,2011)、出願人は、最初にiPSCを胚体内胚葉(DE)の特定化を介して前腸スフェロイドに分化させた。前腸スフェロイドを同じウェルで増殖した付着細胞と混合し、混合物をMatrigelに包埋した。RAは環境依存プロセスを介した多様な系統に対する既知の指定子であるので、出願人は、肝細胞培養培地(HCM)中で培養されて、図1、Aに示すように20日目に確立されたオルガノイドに特徴付けられる肝細胞成熟プロセスの前の初期RAシグナル伝達の持続時間を設定した。培養培地中の最高濃度のアルブミンは、0〜5日の様々な期間のRAの中で4日間のRA曝露で観察されたので、確立されたオルガノイドのさらなる特徴付けをRA曝露0〜4日間の2つの条件間で比較した。RAの処理されたウェルでは、未処理のものと比較してオルガノイドの数は1.8倍増加し、サイズはRA処理後に1.5倍に増加した(図1、B、C、およびD)。アルブミン分泌は、未処理(0.5〜3.5μg/ml、図1、E)と比較した場合、RA処理群(3.0〜6.5μg/ml)において2倍増加した。注目すべきことに、アルブミン分泌が、アルブミンが最初に検出された20日後から40日以上維持された(データは示さず)。興味深いことに、内部管腔構造は95%のRA処理オルガノイドに現れたが、未処理オルガノイドの12%にしか検出されず、オルガノイドの内腔化はRAシグナル伝達に依存することを示した(図1、F)。
興味深いことに、RA処理HLOの単細胞RNA配列決定(scRNA−seq)分析は、星状細胞マーカー(ACTA2、DES、PDGFRB)、クッパー細胞マーカー(CD68、IRF7)、および内皮細胞マーカー(OIT3、DPP4、C1QTNF1;図1、H)によって証明されるように、星状細胞、肝内在性マクロファージ、クッパー細胞、および内皮細胞に特異的な発現痕跡を示した(Bahar Halpern et al.,2017、El Taghdouini et al.,2015、van de Garde et al.,2016)。星状細胞およびクッパー細胞の存在を確認するために、出願人は、上皮細胞マーカーEpCAM、星状細胞マーカーCD166/ALCAM、ならびにクッパー細胞マーカーCD68およびF4/80を用いてFACSによる定量分析を行った(Yanagimachi et al.,2013)。EpCAM+細胞の頻度はHLOの78.85±7.35%であったが、EpCAM−CD166+、EpCAM−CD68+、およびEpCAM−F4/80+発現細胞はそれぞれ32.4±1.2%、1.69±0.3%、および1.68±0.2%であった(図1、I)。免疫組織化学により、CD68細胞の発現もHLOで検出され、CD68発現は内腔側の細胞に局在していた(図1、J)。ヒト肝星状細胞の新規マーカーであり、げっ歯類における星状細胞の分化転換のプロセスに必要であると報告されているビメンチンおよびGFAP(Geerts et al.,2001、Kordes et al.,2014)は両方ともHLOで検出され、同じ細胞中で同時発現し(図1、J)、これは星状細胞がHLOに存在することを示している。
現在、遊離脂肪酸(FFA)がインビトロの脂肪毒性肝細胞モデルを確立するための開始因子として広く使用されているが、しかしながら、脂肪蓄積が唯一の表現型である(Kanuri and Bergheim,2013)。肝細胞とクッパー細胞の代替共培養モデルは、ILファミリーサイトカイン、マクロファージ関連サイトカイン、およびMMP関連サイトカインの発現増加などの炎症反応を示したが、その後の線維症は示さなかった(Hassan et al.,2014)。包括的なトランスクリプトーム解析によって示される脂質代謝機能関連遺伝子を有する肝細胞、星状細胞およびクッパー様細胞の存在を考えると、出願人は、FFA曝露が自然に炎症反応および結果として生じるHLOにおける線維性反応を引き起こすと仮定した。この仮説を検証するために、出願人はFFAを用いて3〜5日間HLOを処理した。出願人は、3日および5日のOA処理HLOを脂肪性肝炎(s)HLOおよび肝硬変(c)HLOとして示した(図4、A)。sHLOは、3日間オレイン酸で処理したHLOであり、脂質蓄積および炎症を示す。cHLOは、5日間オレイン酸で処理されたHLOであり、脂質蓄積および炎症に加えて線維症(HLOは硬化する)を示す。最初に、脂質蓄積に関して、オレイン酸(OA、18:1 n9)、リノール酸(LA、18:2)、パルミチン酸(PA、16:0)、およびステアリン酸(SA、18:0)を含む複数のFFAの効果を比較するため、生細胞イメージングを、FFA曝露の3日後に脂質色素BODIPYを用いて実施した。図7が示すように、OAは肝細胞様細胞における脂質蓄積の誘導に最も効果的であり、SAは最も効果が低かった。OAが大量の脂質蓄積を引き起こすことを考慮して、sHLOにおける炎症反応を引き起こすためにOAの濃度を変えた(0、200、400、および800μM)。生細胞イメージングおよびその後の定量化により、脂質蓄積が用量依存的にsHLOにおいて上昇したことが示された(18倍まで;図4、BおよびC)。脂質蓄積の増加に加えて、脂肪滴のサイズは拡大した(図4、B)。肝細胞バルーニング(拡大)は、非アルコール性脂肪性肝炎(NASH)活性(NASスコアリング)を決定するための病理学的等級付け指標の1つであり、これは、細胞膜のライブイメージングにより800μMのOA処理sHLOにおいて確認された(図4B)。肝臓に蓄積された脂質の主成分であるトリグリセリドも、800μMの処理されたsHLOにおいて検出されたが、未処理のsHLOでは検出されなかった(図4、D)。より重要なことに、培養上清のELISAは、未処理のものと比較した場合、IL−6が800μMのOA処理sHLO培地中で2.2倍分泌されたことを示した(図4、E)。IL−8およびTNF−アルファもまた、200μMまたは800μMのOA処理条件下でアップレギュレートされた(それぞれ10倍および2倍の増加;図4、F)。さらに、OA処理sHLO馴化培地を用いて、トランスウェルで培養してから24時間後にTHP1の遊走を評価し、THP−1遊走細胞は、OA処理sHLO中で上昇し、これは、FFA処理が、おそらく肝細胞様細胞にストレスを引き起こすことによってsHLOにおいて炎症反応を自然に誘発したことを示唆している(最大2倍:図4、Gおよび図8)。これらの炎症および線維症の特徴がHLOに特異的であるかどうかを確認するために、出願人はE−cad mRuby胚性幹(ES)細胞由来のオルガノイドから上皮マーカーE−cadを単離し、E−cad陽性または陰性細胞のいずれかからスフェロイドを再構築し、そしてIL6およびP3NPについてのELISAならびにIL8およびTNF−αについてのRNA発現に適用した。図9が示すように、E−cad陽性細胞も陰性細胞由来スフェロイドも、P3NPおよびIL−6分泌産生を引き起こさず、炎症マーカーの遺伝子発現を過剰発現しなかった。出願人はまた、ヒト肝細胞細胞株HepG2、マクロファージ細胞株THP−1、および肝星状細胞LX−2を用いて同じ実験を試験し、そしてE−cad陽性および陰性細胞の同様の結果を有した。結果は、肝臓オルガノイドが炎症および線維症反応に対するFFA治療にのみ反応することを示した。
AFMによる線維症のハイスループット定量
蓄積された証拠は、肝臓の硬化が肝線維症の重症度とよく相関していることを示し(Yoneda et al.,2008)、それによりHLOの硬化を測定することはHLOの線維症の重症度を評価する可能性がある。平滑筋アクチン免疫染色の出願人の予備的定性分析は、OA曝露による用量依存性線維症進行を示す(図2、H、I、およびJ)。スクリーニング可能なフォーマットにおいてより定量的な洞察を得るために、出願人はこのように、HLO線維症が原子間力顕微鏡(AFM)を用いた微小押し込み(micro−indentation)を用いて用量依存的に硬化を増大させることができるかどうかを生きた状態で評価した。図4のAに示すように、各単一HLOの上部領域(25×25μm四方の14×14マトリックス)をAFMカンチレバーで走査し、これは、HLOのトポグラフィー的および機械的情報の空間マッピングを提供することができる。単一HLOの計算されたヤング率(E、kPa)の代表的なヒストグラムは、明らかにガウス様分布を示し、そのピーク値および幅は、OA濃度に従って増加した(図4、B;0、200、400、800μM)。HLOから決定されたヤング率は、箱ひげ図によるドットプロットによって要約され(図4、C)、これは、HLOの硬さが0から800μM OAまで徐々に上昇すること、すなわち中央値の緩やかなシフトおよび硬化範囲の拡大が、OA濃度の上昇に従って観察された。エメディアン値は、0、200、400、および800μMのOA処理オルガノイドについて、それぞれ1.2kPa、1.6kPa、2.4kPa、および2.8kPaであった。さらに、90パーセンタイルと10パーセンタイルとの間の差(ΔP90−10)は、未処理オルガノイドについては3.2kPaであり、800μM OA処理オルガノイドについては7.0kPaであった。図3、Fが示すように、TNF−アルファおよびIL−8もまた、NAFLD患者における線維症の重症度と相関することが知られているOA添加によってアップレギュレートされた(Ajmera et al.,2017)。これらの結果は、HLOの硬化が線維症の重症度について定量化可能な測定値となり得、したがって線維症のハイスループットスクリーニングに潜在的に適用され得ることを示す。
多能性幹細胞由来の線維症および肝硬変に関するモデルの臨床的関連性は、患者における遺伝的影響の未確定の性質のために不明である。線維症の素因はおそらくこのモデルでは捉えられないであろう。したがって、出願人は、健常人(317D6)、NAFLD/NASH患者(NAFLD150、77、および27)およびウォルマン病患者(WD90、91、および92)を含むiPSC由来の先天性脂肪性肝炎患者を評価することによって臨床表現型に対するアッセイの忠実度を調べた(図5、A)。具体的には、出願人は、致命的な脂肪性肝炎を伴う単一遺伝性疾患である3つのウォルマン病患者特異的iPSC株を確立し、特に臨床診断当時の酵素活性との顕著な相関を伴う正常iPSC株由来オルガノイドと比較して有意な硬さ増加を確認した(図5、B)。
第二に、出願人は脂肪性肝炎の進行に対する多遺伝子効果を評価するためにこの新しいオルガノイドモデルを拡張した。大規模なGWASにより、肝臓トリグリセリド含有量(HTGC)の独立した決定因子であることが繰り返し示されている、3つのタンパク質コーディング配列変異体PNPLA3(パタチン様ホスホリパーゼ3)p.I148M、TM6SF2(膜貫通型6スーパーファミリーメンバー2)p.E167K、およびGCKR(グルコキナーゼ調節蛋白質)p.P446L(Xu et al.,2015)(Zain et al.,2015)(図6、A)。したがって、出願人は大規模セルバンクにアプローチし、以下に示す、報告された効果の大きさβ(SNP)(Stender et al.,2017)、加重投与量を乗算する標準化HTGCおける対立遺伝子ごとの変化、の合計により、2504個の細胞株を多遺伝子スコア(図6、B)でマッピングした:Σ{β(SNP)×(リスク対立遺伝子の投与量)}。3つの異なる閾値を有するiPSC株の獲得後(図6、BおよびC)、出願人は7つのiPSCオルガノイドを生成し、そして表現型に対する多遺伝子性の影響を推定するために脂肪肝炎を誘発した。驚くべきことに、出願人は、「脂肪症」(ライブイメージングによる、図6、DおよびE)と「炎症」(qRT−PCR、図6、F)との顕著な相関関係を見出したが、「線維症」(AFMに基づく硬化測定、図6、G)については多遺伝子性スコアとの相関関係は見出せなかった。出願人はまた、細胞株間でOCA応答が異なり、OCAに対する応答はSNP数に依存することを見出した(図6H)。
ヒトPSCからの多細胞肝臓オルガノイド
一連の最近の研究は、内皮細胞(Takebe et al.,2013)、間葉系細胞(Takebe et al.,2015)、および神経堤細胞(Workman et al.,2017)を実験的に組み合わせることによって支持系統の内胚葉由来オルガノイドへの統合の成功を報告した。RA脈動に基づく方法は、細胞を持続的な細胞極性で多様化するように自然に関与させ、そして合理的な費用で著しく再現性がありそして拡張性がある。支持系統の正確な性質が発生的に関連するものであるか肝臓特異的なものであるかにかかわらず、間質集団はLPSおよび脂肪酸を含む既知の線維症誘発物質に対して完全に反応性であり、多細胞および複雑な病理学をモデル化するための新しい方法を開く。
将来のスクリーニングの観点からは、単一のオルガノイドベースの肝臓測定は、その堅牢性、正規化、および比較的単純さのために非常に魅力的な読み出し情報である。例えば、オルガノイドのライブ蛍光イメージング分析に基づいて、嚢胞性線維症患者の腸オルガノイドについて機能的膨潤アッセイが確立され、効果的な薬物選択が実証された(Saini,2016)。iPSCからの単一の肝臓オルガノイドのライブ硬さ評価は、線維症の重症度を予測するための効果的な方法である。脂肪肝疾患患者では、肝臓の硬化の測定値と線維症の病期との間に有意な相関関係がエラストグラフィによって臨床的に報告されている(Yoneda et al.,2008)。さらに、超音波検査で定義されたNAFLDを有する対象のサブグループにおいて、肝臓の硬化の増加と真性糖尿病(DM)の存在および/またはより高いインスリン抵抗性との間の強い関連性が観察された(Koehler et al.,2016)。興味深いことに、ヒト肝臓オルガノイド(HLO)硬化は、炎症性サイトカイン産生および線維芽細胞増殖を伴って、LPSおよびFFAの両方に比例して増加した。多数の上皮臓器線維症が多様な病理学的メカニズムを介して同様の表現型を共有することを考慮すると、オルガノイドに基づく硬さ検出アッセイは、肺、腎臓、心臓および腸オルガノイドを用いて線維症を分析するために使用できる。
患者特異的なiPSC由来のオルガノイドは、個別化された薬効と上皮反応を予測するために使用されるかもしれない。ヒトiPSCは、健康な人と病気の人の両方から樹立することができる。集団iPSCパネルの確立と並行して、患者のiPS細胞の使用は、脂肪毒性、薬効および安全性に関する個人間の差異をモデル化することが期待される(Warren et al.,2017a、Warren et al.,2017b)。そうすることで、肝臓オルガノイドベースのプラットフォームを用いた表現型スクリーニングは、障害に対する非常に効果的な介入の個別選択を促進するだろう。例えば、栄養補給は個人に応じて変更され得るので、開示されたシステムは投与前に栄養関連状態を反映するための非常に適合性のあるアッセイであり得る。具体的には、肝脂肪症および線維症(PNALD)への起こり得る進行を最小限に抑える目的で、患者特異的iPSC由来オルガノイドを使用して、各患者に対してPN製剤をカスタマイズすることができる。実際、診療所では、PN製剤はしばしばカスタマイズされており(Mercaldi et al.,2012)、なぜなら、肥満で体液制限を必要とし、肝/腎機能障害を示すことが多い、重症患者のカロリー、アミノ酸、および電解質のニーズを市販の溶液は満たさないからである(Boullata et al.,2014)。栄養上の必要性を超えて、PN製品に関連して起こり得る悪影響の低減についての洞察を得ることが緊急の必要性である。したがって、肝臓オルガノイドは、特にPNALDリスク評価についての安全上の懸念を評価するのに有用であり、各患者に対するPN製剤のカスタマイズ戦略を容易にする。
hPSC維持この研究で使用されたTkDA3ヒトiPSCクローンは、K.EtoおよびH.Nakauchiの好意により提供された。ヒトiPSC株を、以前に記載されたように維持した(Takebe et al.,2015;Takebe et al.,2014)。5% CO2、95%空気中、1/30希釈で37℃のMatrigel(Corning Inc.、NY、USA)でコーティングしたプレート上で、未分化hiPSCをmTeSR1培地中でフィーダー不含条件下で維持した(StemCell technologies、Vancouver、Canada)。
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Claims (13)
- 肝臓オルガノイドを遊離脂肪酸(FFA)組成物と接触させるステップを含み、前記FFA組成物がオレイン酸、リノール酸、パルミチン酸、またはそれらの組み合わせ、好ましくはオレイン酸を含む、脂肪毒性オルガノイドモデルの作製方法。
- 前記脂肪毒性オルガノイドモデルが脂肪肝疾患のモデルである、請求項1に記載の方法。
- 前記脂肪毒性オルガノイドモデルが脂肪性肝炎のモデルである、請求項1に記載の方法。
- 前記脂肪毒性オルガノイドモデルが肝硬変モデルである、請求項1に記載の方法。
- 前記脂肪毒性オルガノイドモデルが、非経口栄養関連肝疾患(PNALD)のモデルである、請求項1に記載の方法。
- 前記脂肪毒性オルガノイドモデルがNAFLDのモデルである、請求項1に記載の方法。
- 前記脂肪毒性オルガノイドモデルが、細胞骨格フィラメント組織崩壊、ROS増加、ミトコンドリア膨化、トリグリセリド蓄積、線維症、肝細胞バルーニング、IL6分泌、脂肪症、炎症、バルーニングおよびマロリー体様(Mallory's body−like)、組織硬化、細胞死、およびそれらの組み合わせを特徴とする、請求項1〜6のいずれかに記載の方法。
- NAFLDおよび/または胆汁うっ滞を含む、肝疾患の治療のための薬物をスクリーニングする方法であって、候補薬物を請求項1に記載の脂肪毒性オルガノイドモデルと接触させるステップを含む方法。
- 前記栄養補助食品/TPNを請求項1に記載の脂肪毒性オルガノイドモデルと接触させるステップを含む、栄養補助食品/TPNの有効性をアッセイする方法。
- 脂肪肝疾患の三次元(3D)肝臓オルガノイドモデルであって、前記オルガノイドが、脂肪症、炎症、バルーニングおよびマロリー体、ROS蓄積およびミトコンドリア過負荷、線維症および組織硬化、ならびに細胞死を特徴とする、オルガノイドモデル。
- 薬物誘発性肝毒性および炎症/線維症の三次元(3D)肝臓オルガノイドモデル。
- 非経口栄養関連肝疾患(PNALD)の三次元(3D)肝臓オルガノイドモデル。
- 前記モデルが、炎症性細胞、例えばT細胞または他の炎症性分泌タンパク質を含まない、請求項1〜12のいずれかに記載の肝臓オルガノイドモデル。
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