JP2018505685A - 動脈内皮細胞集団の生成 - Google Patents
動脈内皮細胞集団の生成 Download PDFInfo
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Abstract
Description
本出願は、その全体が参照として本明細書に組み込まれている2015年2月20日出願の米国仮特許出願第62/118,553号に付与された優先権を主張する。
連邦政府資金による研究または開発に関する記述
本発明は、米国国立衛生研究所(国立先進トランスレーショナル科学センター)より授与されたUH2−TR000506に基づく政府支援を受けてなされた。政府は、本発明において一定の権利を有する。
多能性幹細胞から動脈内皮細胞が生成できれば、それは、心血管系疾患を治療すると予想される疾患または状態に対する治療の開発にとって極めて有望である。ただし、一次動脈内皮細胞は培養中に脱分化するので、動脈内皮の開発が課題である。例えば、Prosperらの米国特許出願公開第2009/0104159号は、動脈分化に対する「潜在能力」を実証する血管内皮細胞を培養および使用する方法について記載している(段落[0136])。さらに、McCloskeyらの米国特許出願公開第2012/0064040号は、胚性幹細胞から内皮細胞を誘導するための、化学的に規定された培養条件について記載する。ただし、この開示もやはり、動脈内皮細胞を胚性幹細胞から分化させる潜在能力を単に実証するに過ぎないようであり、その成果は極めて低い。
ヒト胚性幹細胞から動脈内皮細胞を誘導する既存のプロトコールは、ほとんど成功していない。したがって、ヒト多能性幹細胞からヒト動脈内皮細胞を生成する効率的で、規定された、スケーラブルな方法に対する必要性が、当技術分野において今なお存在する。
いくつかのケースでは、骨形成タンパク質(BMP)、アクチビンA、およびWnt/β−カテニンシグナル伝達の活性化因子を含む、血清非含有、アルブミン非含有の化学的に規定された細胞培養培地中で、ヒト多能性幹細胞を約2日の期間にわたり培養して、中胚葉細胞を含む細胞集団を得ることによって、中胚葉細胞が得られる。中胚葉細胞は、Brachyury(T)、EMOS、FOXA2、MIXL1、MSX1、およびMSX2からなる群から選択される1つまたは複数の中胚葉マーカーを発現することができる。多能性幹細胞は、ヒト胚性幹細胞またはヒト誘導多能性幹細胞であり得る。Wnt/β−カテニンシグナル伝達の活性化因子は、Gsk3阻害剤であり得る。Gsk3阻害剤は、CHIR99021、CHIR98014、BIO−アセトキシム、BIO、LiCl、SB216763、SB415286、ARA014418、1−アザケンパウロン、およびビス−7−インドリルマレイミドからなる群から選択され得る。Notch作動薬は、レスベラトロール(3,4’,5−トリヒドロキシスチルベン)、バルプロ酸、およびスベロイルビスヒドロキサム酸からなる群から選択され得る。TGF−ベータ阻害剤は、SB431542であり得る。イノシトールモノホスファターゼの阻害剤は、L−690,330であり得る。
別の態様では、本明細書に提示する方法に従って得られた、実質的に純粋な、多能性幹細胞に由来する動脈内皮細胞の単離された集団が本明細書に提示される。単離された集団は、少なくとも90%の動脈内皮細胞、または少なくとも99%の動脈内皮細胞を含み得る。
本発明は、いくつかの特定の因子を組み合わせることにより、単一の因子と比較して、動脈内皮細胞の分化が大幅に改善したという、本発明者らの発見に少なくとも部分的に基づく。本明細書に記載するように、本発明者らは、ある特定の因子(インスリン、TGFβ、およびPDGF)が動脈内皮の分化を阻害することをさらに発見した。
方法
代表的な実施形態では、本明細書に提示する方法は、中胚葉幹細胞を動脈内皮細胞に分化させるステップを含む。本明細書で使用する場合、用語「動脈内皮細胞」(AEC)とは、本明細書に提示する方法に従って得られた動脈血管内皮系列の細胞を意味する。本発明のAECは、動脈内皮細胞マーカー、例えばエフリンB2、DLL4、Hey−2、ジャギド−1、およびジャギド−2等の高レベルの発現により特徴付けられる。AECは、白血球接着が低く、NO生成および酸素消費量、剪断応力に対する応答、ならびにin vitroおよびin vivoでの血管ネットワーク形成能力がより高い一方、そのようなネットワークにおいて動脈マーカーの発現が維持されることによっても特徴付けられる。AECは、本明細書に記載するように、細胞のin vitroでの特徴的な発現プロファイルおよび機能的特性に基づいて、内皮細胞(EC)、静脈内皮細胞、および内皮前駆細胞を含むその他の細胞型から区別可能である。
本発明の方法で使用するのに適するTGFβ受容体阻害剤には、SB−431542、SB−525334、A83−01、LY2157299、LY210976、GW788388、RepSox、SB−505124、D4476、GW788388、SD208、およびEW−7197が非限定的に含まれる。好ましくは、TGF−ベータシグナル伝達の阻害剤は、SB431542、内因性アクチビンの小分子阻害剤、およびI型受容体(TGFβ受容体I)である(Inman et al., Mol Pharmacol. 62(1):65-74 (2002))。
好ましくは、中胚葉細胞は、得られた細胞集団の少なくとも約80%(例えば、少なくとも80%、85%、90%、95%、99%)が動脈内皮細胞となるまでAEC分化培地中で培養される。動脈内皮細胞は、下記の発現プロファイル:CD31+/CD144+/CD41-/CD45-を特徴的に有する。
さらなる態様では、したがって、本発明は、細胞移植、細胞の補充、および細胞または組織の交換、および脈管形成強化のための方法および組成物を提供する。本方法は、本明細書に提示する方法に従い誘導される、治療有効量の動脈内皮細胞を、それを必要としている対象に提供するステップを含み得、それにより動脈内皮細胞による対象の治療を提供する。細胞または組織の交換、および脈管形成の改善を必要とする障害として、心筋および末梢血管の虚血、その他の末梢動脈疾患、心筋梗塞(MI)、脳卒中、および糖尿病性神経障害、ならびに罹患した個体が血管形成の再生医療から利益を得るような任意のその他の障害または疾患が、非限定的に挙げられる。本発明による好ましい個々の対象として、ヒトおよび非ヒト霊長類、ならびにイヌ、ネコ、ヒツジ、ブタ、ウマ、およびウシを非限定的に含む、哺乳動物が挙げられる。いくつかのケースでは、実質的に純粋な動脈内皮細胞の集団が、治療を必要とする対象の多能性細胞(例えば、誘導多能性幹細胞)を使用して得られる。ただし、実質的に純粋な動脈内皮細胞の集団は、好ましくは同系または同種のドナーの多能性幹細胞を使用しても得ることができる。それほど好ましくはないが、異種のドナーも使用される。
別の態様では、本明細書に提示する方法に従って得られたAECは、一酸化窒素(NO)の生成が、対象に対して治療または予防上の利益をもたらすような方法で有用である。例えば、AECを対象に投与する方法が、NOを対象に提供する方法として本明細書に提示され、これにより、AECの投与がアテローム性動脈硬化症を治療または予防する、DNA損傷を低減する、および/または血管機能が改善するように平滑筋細胞を弛緩させる。
別の態様では、AECの調製物が本明細書に提示される。例えば、AEC、実質的に精製されたAECの集団、AECを含む医薬製剤、および凍結保存されたAECの調製物が、本明細書に提示される。本明細書に記載するAECは、その天然の環境に見出される少なくとも1つのタンパク質、分子、またはその他の不純物について実質的に非含有であり得る(例えば、「単離された」)。AECは、ヒトを含む哺乳動物AECであり得る。本発明は、ヒトAEC、実質的に精製されたヒトAECの集団、ヒトAECを含む医薬製剤、および凍結保存されたヒトAECの調製物も提供する。調製物は、ヒト胚性幹細胞由来のAEC、ヒトiPS細胞由来のAECを含む調製物、および分化後の多能性幹細胞由来のAECを含む、実質的に精製された(非AECに関して)調製物であり得る。
本発明は、ヒト多能性幹細胞をAECに分化させるキットであって、(i)ヒト多能性幹細胞を中胚葉細胞に分化させるのに適する第1の培養培地と、(ii)多能性幹細胞由来の中胚葉細胞を動脈内皮細胞に分化させるのに適する第2の培養培地と、(iii)ヒト多能性幹細胞をCD31+/CD144+/CD41-/CD45-動脈内皮細胞に分化させる方法であって、第1の培養培地および第2の培養培地を利用する方法について記載する説明書とを含むキットも提供する。
別途規定しない限り、本明細書で使用するすべての技術用語および科学用語は、本発明が属する当業者により一般的に理解される意味と同一の意味を有する。本明細書に記載する方法および材料と類似する、または同等の任意の方法および材料が、本発明の実践または試験に使用できるが、好ましい方法および材料が本明細書に記載されている。
本明細書で使用する場合、「有効量」とは、本発明による規定された細胞の効果を惹起するのに十分な薬剤の量を意味する。
本明細書で使用する場合、「約」とは、記載された濃度範囲、密度、温度、またはタイムフレームの5%以内を意味する。
本発明は、下記の非限定的な実施例について検討すれば、よりよい理解が得られる。開示する方法は、多能性幹細胞に一般的に適するものと特に考えられる。本明細書に開示するすべての文書および特許は、その全体が記載されているかのように、本明細書に参照として組み込まれる。
動脈の分化を調べるために、血清およびウシ血清アルブミンの両方を欠いている規定された培養培地を使用して、内皮細胞分化プロトコールを策定した。まず、BMP4、アクチビン−A、およびCHIR99021が補充された培養培地(E8BAC培地)中で、異物を含まない多能性幹細胞を、中胚葉細胞に2日間分化させた。次いで、中胚葉細胞を、FGF2、VEGFA、およびBMP4でさらに3日間処理し、70%のCD31+/CD34+内皮細胞集団を得た(図2A〜B)。インスリンが、この中胚葉から内皮細胞への分化培地に含まれた。内皮細胞の生死を、NANOGおよびOCT4の下方制御(図2C)、KDR/VEGFR2の上方制御、CD144の発現(CDH5/VE−カドヘリン)(図2D)、LDLのインターナリゼーション(図2E)、ならびにin vitroおよびin vivoでの毛細血管ネットワークの形成(図2F〜G)によりさらに確認した。このプロトコールを用いて、本発明者らは、完全に規定された培養条件下で、個々の培地構成成分の効果を調べることができた(図2H)。
本発明者らは、EFNB2−tdTomato/EPHB4−EGFPデュアルレポーター細胞株を使用して、単一細胞RNA−Seq分析により識別された個々の増殖因子関連遺伝子の機能を試験した。これまでに記載されているその役割と整合して、VEGFA、WNT3A、およびRESV(Notch作動薬)は、すべて、動脈の特異化の向上を促進した(図11)。
単一細胞RNA−seq用のマウス内皮細胞の単離:24匹のE11.5マウス(CD−1バックグラウンド)胚を採取した。頭部、尾部、四肢、内臓、および体節を取り出した。大動脈−性腺−中腎(AGM)組織を、2mg/mlのコラゲナーゼIV型(Life technologies、カタログ番号17104−019)、および0.25mg/mlのディスパーゼ(Life technologies、カタログ番号17105−041)を含む溶液中で、酵素が組織に浸透するように、氷上で15分間インキュベートした。次いで、酵素を含む組織を、37℃で10分間インキュベートした。酵素を2%のFBS−HBSSで中和し、上下にピペット吸引して細胞をさらに解離させた。細胞を免疫染色し、CD31+CD144+CD41-CD45-内皮細胞をフローサイトメトリーによりソーティングした。CD41およびCD45を使用して造血幹細胞を枯渇させた。
一次AEC(「pAEC」;14週齢のヒト胎児背側大動脈から新たに単離した)では、cDNAを調製するために、smarter−seq2プロトコールを、Fluidigm C1(商標)単一細胞自動調製システムに適用した。Smarter−seq2は、cDNA収率および配列決定感度41を改善することが示されており、したがって、RNAの質が比較的低いサンプルに適する。合計48個の細胞について配列決定を行い、さらなる分析に使用した。
階層的クラスタリング:単一細胞RNA−seqデータ(TPM)をRSEMから生成した。遺伝子毎に、log2 TPMを平均値0およびばらつき1でzスコアにスケール化した。対数化する前に、1未満のTPMを1とした。細胞間のユークリッド距離を用いて階層的クラスタリングを実施した(図1)。
SINGuLAR分析ツールセット2.1によるデータ分析:単一細胞RNA−seqデータ(TPM)をSINGuLAR分析ツールセット2.1に充填した。外れ値を、「identifyOutliers()」コマンドにより除去した。次いで、サンプルの動脈および静脈マーカーを、「autoAnalysis()」コマンドにより分析した。その結果、図1CのPCAプロットを自動的に生成した。AECデータのヒートマップ(図10)も、SINGuLARの「autoAnalysis()」により生成した。
H1ES細胞上での遺伝子標的化:EFNB2標的化ベクターの5’および3’ホモロジーアームを、IDT(gBlock)により、Sal IおよびBamH I(5’アーム)制限部位、Bmt IおよびMlu I(3’アーム)制限部位を導入しながら合成して、標的化ベクターへのサブクローニングを促進した。EPHB4標的化ベクターの5’および3’ホモロジーアームを、BAC(細菌人工染色体)からPCR増幅した。
サザンブロット:PCR DIGプローブ合成キット(Roche、カタログ番号11 636 090 910)を使用してプローブを合成した。Rocheから入手したフィルターハイブリダイゼーションに関するDIGアプリケーションマニュアルに従ってサザンブロットを実施した。
機能的アッセイの一部について、AECをCD144マイクロビーズ(Miltenyi Biotec、カタログ番号130−097−857)により精製した。最適化後(図9)、FVIR(E5+100ng/mlのFGF、50ng/mlのVEGF、10μMのSB431542、5μMのRESV)、またはFVIR+Ins(FVIR培地+10μg/mlのインスリン)培地中、フィブロネクチンまたはビトロネクチンコーティング培養皿上でAECを維持した。
MATRIGEL(登録商標)カプセル化アッセイ:1.5×103個の内皮細胞/μl、および0.75×103個の周皮細胞/μl(ScienCell、カタログ番号1200)を、6.5mg/mlのMatrigel中でカプセル化した。10μLのMatrigel/細胞溶液を24ウェルプレートの中央にスポットし、凝固させるために37℃で5分間インキュベートした。次いで、E7V培地を適用した。免疫染色を4日目に実施し、そしてNikon共焦点顕微鏡を使用して構造を画像化した。
酸素誘発性網膜症モデル:実験を、UW−Madisonの眼科学およびビジュアルサイエンス(Ophthalmology and Visual Science)のIRBから承認を得て実施した。これまでの記載の通り21、C57/BL6野生型マウスで酸素誘発性網膜症を誘発した。要するに、出生後7日目に、マウスを75%の酸素に5日間曝露した。出生後12日目に、マウスを室内空気に戻し、5×104個の細胞を含有する1μlを硝子体内注射した。リン酸緩衝化生理食塩水(PBS)をビヒクルとして使用し、対照として注射した。5日後に網膜を採取し、免疫染色を実施した。
FVIR+Ins培地は内皮細胞の伸長を促進したので、24時間剪断応力応答実験の前および間に、E7V培地を、「5因子」AECを培養するのに使用した。
Claims (24)
- 動脈内皮細胞を得る方法であって、
インスリンを実質的に含まず、線維芽細胞増殖因子(FGF)、血管内皮増殖因子(VEGF)、ならびにNotch作動薬、TGF−ベータ阻害剤、およびイノシトールモノホスファターゼの阻害剤のうちの少なくとも1つを含む、血清非含有、アルブミン非含有の化学的に規定された培養培地中で、中胚葉細胞を培養することにより、動脈内皮細胞を含む細胞集団を得るステップ
を含み、
該集団の動脈内皮細胞が、エフリンB2(EFNB2)、ニューロピリン1(NRP−1)、デルタ様4(DLL4)、CD44、CXCR4/CD184、Gap結合タンパク質α−4(GJA4)、Hey1、ジャギド−1(JAG1)、Notch1、およびNotch4からなる群から選択される1つまたは複数のマーカーを発現する、方法。 - 細胞集団が、少なくとも80%の動脈内皮細胞を含む、請求項1に記載の方法。
- 血清非含有、アルブミン非含有の化学的に規定された培養培地が、FGF、VEGF、Notch作動薬、TGF−ベータ阻害剤、およびイノシトールモノホスファターゼの阻害剤を含む、請求項1に記載の方法。
- 中胚葉細胞が、Brachyury(T)、EMOS、FOXA2、MIXL1、MSX1、およびMSX2からなる群から選択される1つまたは複数の中胚葉マーカーを発現する、請求項1に記載の方法。
- 骨形成タンパク質(BMP)、アクチビンA、およびWnt/β−カテニンシグナル伝達の活性化因子を含む、血清非含有、アルブミン非含有の化学的に規定された細胞培養培地中で、ヒト多能性幹細胞を約2日の期間わたり培養して、中胚葉細胞を含む細胞集団を得ることによって、中胚葉細胞が得られる、請求項1に記載の方法。
- 中胚葉細胞が、Brachyury(T)、EMOS、FOXA2、MIXL1、MSX1、およびMSX2からなる群から選択される1つまたは複数の中胚葉マーカーを発現する、請求項5に記載の方法。
- 多能性幹細胞が、ヒト胚性幹細胞またはヒト誘導多能性幹細胞である、請求項5に記載の方法。
- Wnt/β−カテニンシグナル伝達の活性化因子が、Gsk3阻害剤である、請求項5に記載の方法。
- Gsk3阻害剤が、CHIR99021、CHIR98014、BIO−アセトキシム、BIO、LiCl、SB216763、SB415286、ARA014418、1−アザケンパウロン、およびビス−7−インドリルマレイミドからなる群から選択される、請求項8に記載の方法。
- Notch作動薬が、レスベラトロール(3,4’,5−トリヒドロキシスチルベン)、バルプロ酸、およびスベロイルビスヒドロキサム酸からなる群から選択される、請求項5に記載の方法。
- TGF−ベータ阻害剤が、SB431542である、請求項5に記載の方法。
- イノシトールモノホスファターゼの阻害剤が、L−690,330である、請求項5に記載の方法。
- 請求項1に記載の方法に従って得られた、実質的に純粋な、動脈内皮細胞の単離された集団。
- 少なくとも90%の動脈内皮細胞を含む、請求項13に記載の単離された集団。
- 少なくとも99%の動脈内皮細胞を含む、請求項13に記載の単離された集団。
- 請求項5の方法に従って得られた、実質的に純粋な、多能性幹細胞に由来する動脈内皮細胞の単離された集団。
- 少なくとも90%の動脈内皮細胞を含む、請求項16に記載の単離された集団。
- 少なくとも99%の動脈内皮細胞を含む、請求項16に記載の単離された集団。
- 薬剤をin vitroでスクリーニングする方法であって、
(a)試験薬剤を、請求項1に記載の方法に従って得られた動脈内皮細胞と接触させるステップと、
(b)接触させた動脈内皮細胞への白血球の接着に対する該薬剤の効果を検出するステップと
を含む方法。 - 検出するステップが、白血球接着アッセイ、RNA配列決定、遺伝子発現プロファイリング、トランスクリプトーム解析、メタボローム解析、レポーターまたはセンサーの検出、タンパク質発現プロファイリング、フェルスター共鳴エネルギー移動(FRET)、メタボリックプロファイリング、およびマイクロダイアリシスからなる群から選択される方法を実施するステップを含む、請求項19に記載の方法。
- 工学的に作出された組織構築物を血管化させる方法であって、請求項1に記載の方法に従って得られた動脈内皮細胞を、工学的に作出された組織構築物と接触させるステップを含む方法。
- 工学的に作出された組織構築物が、血管平滑筋細胞を含む、請求項21に記載の方法。
- 動脈内皮細胞を得るためのキットであって、(i)中胚葉細胞を動脈内皮細胞に分化させるのに適する血清非含有、アルブミン非含有の化学的に規定された培養培地であって、インスリンを実質的に含まず、線維芽細胞増殖因子(FGF)、血管内皮増殖因子(VEGF)、ならびにNotch作動薬、TGF−ベータ阻害剤、およびイノシトールモノホスファターゼの阻害剤のうちの少なくとも1つを含む、培養培地と、(ii)該培養培地を利用する、中胚葉細胞を動脈内皮細胞に分化させる方法を説明する説明書とを含む、キット。
- (a)ヒト多能性幹細胞を中胚葉細胞に分化させるのに適する、血清非含有、アルブミン非含有の化学的に規定された培養培地であって、BMP、アクチビンA、およびWnt/β−カテニンシグナル伝達の活性化因子を含む、培養培地と、
(b)ヒト多能性幹細胞を動脈内皮細胞に分化させる方法であって、(a)の培養培地を利用する方法について記載する説明書と
をさらに含む、請求項23のキット。
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