JP2020503878A - 多能性幹細胞の培養のための組成物 - Google Patents
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Abstract
Description
ニューレグリン1; HRG-β、HRG-α、Neu分化因子(NDF)、アセチルコリン受容体誘導活性(ARIA)、グリア成長因子2(GGF2)、並びに感覚及び運動ニューロン由来因子(SMDF)を含むがこれに限定されないニューレグリン1のスプライスバリアント及びアイソフォーム;
ニューレグリン2; NRG2-β;エピレグリン;及びビレグリンを含むがこれに限定されないニューレグリン2のスプライスバリアント及びアイソフォームが挙げられる。
1. 分析しようとする因子を添加することによって培地を調製する工程と;
2. 前記培地中でヒト胚を培養する工程と;
3. 前記胚を、前記因子を含まない培地中で又は前記因子の阻害剤若しくは活性化剤を含有する培地中で培養した胚と比較する工程と;
4. 前記因子が前記胚の培養に必須かどうかを決定する工程と
を含む。
材料及び方法
ヒト胚盤胞トランスクリプトームデータセットの生成
推定上の別個の成長因子又はシグナル伝達経路を同定するために、ヒト胚から単離した単一細胞の分析から最近生成されたRNA配列決定(RNA-seq)データセットを使用した(Blakeleyら、2015年、Development、142:3151〜3165頁)。簡潔には、胚盤胞段階のヒト胚(E6-E7)をレーザー顕微解剖して、大部分の壁栄養外胚葉を内部細胞塊(ICM)及び極栄養外胚葉(極TE)から分離した。ICM及び極TEを、0.05%トリプシン/EDTA(Invitrogen)中で5〜10分間インキュベートし、30μm内径割球生検ピペット(Research Instruments)を使用して単一細胞を単離した。RNAを単一細胞から抽出し、Illumina Sequencing-HV(Clontech Laboratories)用のSMARTer Ultra Low RNA Kitを使用してDNA合成処理を行った。Covaris S2を、10%デューティ、強度5、連射サイクル200に設定を修正して使用して、cDNAを2分間剪断した。
ヒト胚は、UK Human Fertilisation and Authority登録番号R0162の元でインフォームドコンセントにより研究計画に贈与された。ストロー中に凍結されているガラス固化胚を、ストローの内容物を液体窒素から直接、解凍溶液(Irvine Scientific Vitrification Thaw Kit)に速やかに移すことによって解凍し、また製造業者の指示通り解凍した。cryopets中に凍結されている胚を、解凍溶液(Irvine Scientific Vitrification Thaw Kit)に移す前に37℃水浴中で3秒間まず解凍した。ガラスアンプル中に凍結されている胚を、液体窒素下でバイアルの上部を除去した後に37℃水浴中で完全に解凍した。内容物をペトリ皿に注ぎ、製造業者の指示通りショ糖溶液(Quinn's Advantage Thaw Kit、Origio)の勾配を通じて胚を移した。
胚を、4%(w/v)パラホルムアルデヒド(PFA)(ThermoFisher Scientific)を用いて回転振とう器上で、4℃で1時間固定し、以前に記載されているようにして分析した(Niakan及びEggan、Developmental Biology、2013年: doi: 10.1016/j.ydbio.2012.12.008)。簡潔には、次いで胚を、カルシウム及びマグネシウムを含まないダルベッコリン酸緩衝食塩水(PBS)(Thermo Fisher Scientific)に希釈した0.1%(v/v)Tween-20(Sigma Aldrich)の数回の洗浄を通じて移して、残留するパラホルムアルデヒドを除去した。胚を、透過化処理のために0.5%(v/v)Tween-20中に20分間置いた。胚を、ブロッキング溶液(0.1%(v/v)Tween-20に希釈した10%ロバ血清)中で、室温で1時間ブロッキングした。胚を、ブロッキング溶液に1:500の濃度の一次抗体中に回転振とう器上で、4℃で終夜置いた。以下の一次抗体を、(全て1:500希釈で)使用した:抗NANOG(AF1997 R&D Systems、REC-RCAB0001P 2B Scientific又はab21624、Abcam)、抗GATA6(SC-9055、Santa Cruz)及び抗SOX17(AF1924、R&D Systems)。次の日、胚を、0.1%(v/v)Tween-20の4回洗浄を通じて移し、次いで、最後の洗浄に30分間置いた。二次抗体(Cy3、FITC又はCy5ロバ抗ウサギ、マウス、又は、ヤギ、Molecular Probes)を1:300の濃度でブロッキング溶液に希釈した。胚を、二次抗体中に回転振とう器上で、室温で1時間置き、0.1%(v/v)Tween-20の4回洗浄を通じて移し、最後の洗浄に30分間置いた。胚を、共焦点画像化のためにボトムディッシュカバーガラス(MatTek)上でDAPI含有Vectashield(Vector Labs):0.1%(v/v)Tween-20の1:3希釈50μL中に置いた。
胚を、ヒト又はマウス胚に対してそれぞれ3μm又は2μmのz-断面厚でLeica SP5倒立共焦点顕微鏡(Leica Microsystems GmbH)で撮像した。MINS 1.3ソフトウェアを使用して核を検出し、区分し、それにより各胚中の細胞数を決定した(http://katlab-tools.org/)、(Louら、Stem Cell Reports 2014年、doi: 10.1016/j.stemcr.2014.01.010)。tif形式の共焦点スタックを、自動化核区分のためにMINSパイプラインにロードした。MINS区分出力を適切な区分について手作業で確認し、有糸分裂核を分析から除去した。データを、GraphPad Prismバージョン6(GraphPad Software、La Jolla、CA)を使用してその後プロットした。
胚培養に対するFGFの効果の調査
ヒト胚を、100ng/ml又は1μg/mlの濃度で、FGF2を補充したヒト胚培養培地中でE2.5又はE5から培養した(図1A)。免疫蛍光分析は、100ng/ml又は1μg/mlのいずれかでE2.5から処理した場合に、無処理の対照と比較してNANOG発現が消失したことを示した(図1)。
トランスクリプトームデータセット(材料及び方法において上記の通り、作製された)に問い合わせを送信し、多能性胚盤葉上層細胞において発現される受容体をコードしている、又はヒト胚において任意の細胞型によって発現されるリガンドをコードしている転写産物を同定した。本発明者らは初期のヒト発達においてインスリン(INSR)及びIGF1(IGF1R)受容体、加えてIGF1リガンド(IGF1)の転写産物の発現を同定した(図2A)。特に、インスリン及びIGF2の発現は、ヒト胚盤胞において検出不能であった。ヒト胚盤胞におけるIGF1及びその同族受容体の転写産物の存在は、これが、調節する興味深い候補になる可能性があることを示した。
ヒトES細胞誘導及び培養系は、細胞増殖のためにMEF支持層をしばしば利用するが、MEFはFGFを分泌するので、本発明者らの要件と適合しなかった。マトリゲル、ラミニン及びビトロネクチンなどの支持マトリックス基質層が、フィーダーフリー培養系においてMEFの代わりに一般的に使用される。しかしながら、マトリゲルは、エンジェルブレスホームスワーン(EHS)肉腫細胞から抽出され、したがって依然としてバッチ間変動性の影響を受け、成長因子低減形態においてもFGFを含めた様々な成長因子の顕著な濃度を保持している。追加の基底膜基質が、ヒト胚に存在するそれらを反映することになるかどうかも不明であった。
材料及び方法
hES及びiPS細胞の培養条件
FGF2を補充したKnockOut Serum Replacement(KOSR)培地(KSR+FGF)における培養のために、細胞を、MEFコーティングした予めゼラチン化した組織培養プレート(Corning)上で20% KOSR及び5ng/ml FGF中で維持した。細胞を、手作業のピッキングによって継代した。
固定化の日に、培地を、多能性細胞株又は分化細胞から除去し、細胞を、カルシウム及びマグネシウムを含まないダルベッコリン酸緩衝食塩水(PBS)(Thermo Fisher Scientific)で洗浄した。細胞を、4%(w/v)パラホルムアルデヒド(PFA)(ThermoFisher Scientific)で1時間固定し、0.5%(w/v)Triton X-100又は0.5%(v/v)Tween-20(Sigma Aldrich)で透過化処理した。PBS(0.05%(v/v)Tween-20又は0.01% Tween-20を含有する)中の2〜10%(v/v)ロバ血清(AbCam)を使用して、非特異的結合を最小化した。以下の一次抗体を、(特に明記しない限り全て1:500希釈で)使用した:抗NANOG(AF1997 R&D Systems; REC-RCAB0001P、2B Scientific; 4903P、Cell Signaling Technologies又はab21624、Abcam)、抗SOX17(AF1924、R&D Systems)、抗TRA-1-81(MAB4381、Millipore;又は560072、BD Biosciences)、抗TUJ1(T2200、Sigma)、抗FOXA2(3143、Cell Signaling)、抗OCT4(sc-5279、Santa Cruz又は2750S、Cell Signaling Technologies)、抗SSEA4(MA1-021、Life Technologies又はMC-813-70、DSHB、1:100)、抗TRA-1-60(MAB4360、Millipore、1:100)、抗CXCR4(MAB173、R&D)、抗デスミン(RB-9014-R7、Neomarkers、1:50)、抗AFP(A0008、Dako)、抗サイトケラチン18(ab668、Abcam)。一次抗体を、PBS(0.05%又は0.01%v/vTween-20を含有する)で洗浄することにより除去した。二次抗体(Alexa Fluor(登録商標)488、Alexa Fluor(登録商標)594、Alexa Fluor(登録商標)555ロバ抗ウサギ、マウス又はヤギ、Molecular Probes)を、1:300の濃度でブロッキング溶液に希釈し、対応するウェルに室温で添加して1時間置いた。次いで、細胞を、0.1%(v/v)Tween-20中で数回洗浄し、最後の洗浄に30分間置いた。DAPIを含有するVectashield(Vector Labs)少量を、画像化前に各ウェルに添加した。
画像は、Leica TCS SP8共焦点顕微鏡又はCell∧Fソフトウェア(Olympus Corporation)と共にオリンパスIX73顕微鏡を使用して取得した。位相差画像は、Leica MC170 H9カメラ(Leica Microsystems GmbH)を用いてLeica DM IL LED顕微鏡で撮像した。
幹細胞誘導前にインキュベーター内で37℃及び5% CO2に予備平衡化した5mg/mL LifeGlobal Protein Supplementを補充したGlobal Media (LifeGlobal)中でE5又はE6ヒト胚盤胞を、まず初めに培養した。Chenら、2009年、Cell Stem Cell: doi: 10.1016/j.stem.2008.12.001に記載のようにして、オリンパスIX73顕微鏡及びSaturn5レーザー(Research Instruments)を使用してE6胚盤胞段階のヒト胚を分散させて、内部細胞塊を単離した。胚を、鉱油を重層したペトリ皿上でHEPESを含む少量のGlobal(登録商標) Total(登録商標)(LGTH、LifeGlobal)中に置いて、顕微操作した。内部細胞塊(ICM)及び極栄養外胚葉を、AI培地中でMEFコーティングした皿にプレーティングした。hES細胞様形態を持つICM生育物を、さらなる増殖のためにMEFコーティングした又はラミニン511のいずれかの皿に手作業で採取した。
ヒトBJ線維芽細胞を、30〜60%コンフルエントになるようにプレーティングして、2日後に形質導入した(6ウェルの、ウェル当たり1×105個細胞)。細胞を、製造業者の指示に従ってCytotune(商標)2.0 Sendai再プログラミングキット(Invitrogen)を使用し、次いで形質導入し、形質導入の6日後、MEFコーティングした皿(KSR+FGF培地)、ビトロネクチンコーティングした皿(TeSR(商標)E8培地)又はラミニン511コーティングした(AI培地)皿に移した。細胞を、この時点で低酸素条件(5% O2、5% CO2、37℃)に移し、誘導の残り期間を低酸素下で培養した。形質導入の18日後、TRA-1-60発現を、製造業者の指示に従って(1:100希釈)Stemgent(登録商標) StainAlive(商標) TRA-1-60抗体(DyLight(商標)488)キットを使用して分析した。多能性ES細胞形態を有するコロニーを、形質導入の22日後に採取して増殖させ、安定なiPS細胞株を樹立した。
稼働細胞バンクのランダムな3本のバイアルの細胞を、ThawStar(登録商標)装置で解凍し、2次元培養系LN511中で、AI培地で別々に培養した。細胞計数及び生存率を、Nucleocounter NC-200(Chemometech)によって測定し、集団倍加数を式1に従って算出した。
表面及び細胞内の多能性関連タンパク質の発現を、MACSQuant(登録商標) Analyzer10血球計算器(Myltenyi Biotec)を使用してフローサイトメトリーによって分析した。単一細胞懸濁液を洗浄し、以下のコンジュゲート抗体: SSEA4(130-098-341、Myltenyi); SSEA3(330306、Biolegend); CD30(550041、BD Biosciences); TRA-1-60(560495、Biolegend); NANOG(560791、BD Biosciences); OCT4(561556、BD Biosciences); SOX2(656108、BD Biosciences)によりBD Pharmigen(商標)Stain Buffer(BD Biosciences)中で染色した。細胞内タンパク質(NANOG、OCT4、SOX2)に対して染色する場合、細胞を、BD Cytofix(商標)固定化緩衝液(BD Biosciences)を使用して固定し、室温で15分間インキュベートした。この後、細胞を、染色に進む前にBD Pharmigen(商標)Stain Buffer中の0.1%(v/v)Triton X-100(Sigma)により室温で15分間透過化処理した。アイソタイプ対照を、各抗体について実行した(SSEA4アイソタイプ、130-104-608、Myltenyi; SSEA3アイソタイプ、400811、Biolegend; CD30アイソタイプ、555749、BD Biosciences; TRA-1-60アイソタイプ、401618、Biolegend; NANOGアイソタイプ、557702、BD Biosciences; OCT3/4アイソタイプ、554680、BD Biosciences; SOX2アイソタイプ、400136、BD Biosciences)。細胞を、Live/Dead(登録商標)判別色素(L23105、ThermoFischer Scientific)で染色し、生存単一細胞集団画分の表現型分析を、フローサイトメトリーによって実行した。アイソタイプ染色を、各分析及び条件に対する陰性対照とした。
Gバンド核形分析に使用するヒトES及びiPS細胞を、懸濁状態で固定した。複数の分裂中期スプレッドをサンプル毎に分析し、染色体数及びGバンドパターンを決定した。
自発的分化のために、AI培地中で培養した細胞をMEF培養培地(10% FBS)に6又は12日間切り替えた。細胞を、3つの胚葉系統について免疫蛍光分析のために次いで固定した。
アクチビン及びIGF1を含む化学的に規定された(Chemically defined)最小培地(AI)は、ヒト多能性を支持するのに十分である
本発明者らは、まず樹立hES細胞を使用して、多能性培養培地においてIGF1がFGF2を置換し得るかどうかを決定した。ラミニン511の生理的関連を考慮して、本発明者らは、特に明記しない限り化学的に規定された(Chemically defined)条件における多能性細胞培養にこの精製された基底膜成分で続けた。生存可能な培養系を樹立する条件を試験するために、H1 hES細胞を、対照mTeSR(商標)1培地、グルタミン補充剤(2mM)を含む基本培地(Advanced-DMEM/F12)、又はグルタミン、12ng/ml FGF2及び10ng/mlアクチビン(Vallierら、2005年、J Cell Sci、118、4495〜4509頁による成長因子濃度)、グルタミン及び17nM IGF1、若しくはグルタミン、10ng/mlアクチビン及び17nM IGF1を補充した基本培地、のいずれかの中で増殖させた(図4A)。アクチビン及びIGF1(今後AI)培地中で培養したhES細胞は、高い核対細胞質比、密集したコロニー及び明瞭なコロニー境界線により対照mTeSR(商標)1処理細胞と似ていた。
これら培養条件のストリンジェントな試験として、本発明者らは、AI培地を使用してヒト胚からhES細胞を直接誘導させ得るかどうかを次に追及した。5日目の胚盤胞を、ヒト胚培養培地中で6日目まで終夜培養した。内部細胞塊(ICM)及び重層している極栄養外胚葉を、壁栄養外胚葉から顕微解剖し、ICM凝集塊をAI培地中にプレーティングした(図8A)。ヒトES細胞様コロニーが、AI培地中へのプレーティング後1週間以内に現れ、ラミニン511上で安定なhES細胞株として維持できた(図8A)。免疫蛍光分析により、hES細胞が、多能性タンパク質NANOG、OCT4、SOX2及びTRA-1-81を発現することが確認された(図8B)。
本発明者らは、AI培地において専ら誘導されたhES細胞が多能性であり、胚葉分化の能力を保持することを確認した。自発的に分化するhES細胞の免疫蛍光分析により、SOX17発現性内胚葉細胞、TUJ1発現性の外胚葉誘導ニューロン、及びデスミン発現性中胚葉細胞に分化するhES細胞の能力を実証した(図10A)。
Claims (23)
- 多能性幹細胞を培養するための細胞培地であって、インシュリン様成長因子1(IGF1)を含み、線維芽細胞成長因子(FGF)及びいかなるFGF受容体活性化剤も含まない若しくは実質的に含まない、かつErbB3リガンドを含まない、化学的に規定された培地(Chemically defined medium)又は最小培地である細胞培地。
- 胚性幹細胞又は誘導多能性細胞を培養するための、請求項1に記載の細胞培地。
- 哺乳動物細胞を培養するための、請求項1又は2のいずれか一項に記載の細胞培地。
- ヒト細胞を培養するための、請求項3に記載の細胞培地。
- 前記IGF1が、配列番号1又は配列番号2の配列を含む、請求項1〜4のいずれか一項に記載の培地。
- TGFβファミリーメンバーをさらに含む、請求項1〜5のいずれか一項に記載の培地。
- 前記TGFβファミリーメンバーが、アクチビンである、請求項6に記載の培地。
- ヒト多能性幹細胞を培養するための細胞培地であって、化学的に規定された培地(Chemically defined medium)又は最小培地であり、基礎培地、IGF1、TGFβファミリーメンバー及びグルタミン補充剤からなる細胞培地。
- 前記TGFβファミリーメンバーが、アクチビンである、請求項8に記載の培地。
- 前記基礎培地が、Advanced DMEM/F12である、請求項8又は9に記載の培地。
- in vitroで多能性幹細胞を培養する方法であって、請求項1〜10のいずれか一項に記載の培地中で前記細胞を培養する工程を含む方法。
- 多能性幹細胞の培養における、請求項1〜10のいずれか一項に記載の培地の使用。
- 前記細胞が、胚性幹細胞又は誘導多能性細胞である、請求項11に記載の方法又は請求項12に記載の使用。
- 前記細胞が、哺乳動物細胞である、請求項13に記載の方法又は使用。
- 前記細胞が、ヒト細胞である、請求項14に記載の方法又は使用。
- 請求項11〜15のいずれか一項に記載の方法によって得られる又は得られた細胞。
- 胚を培養するための培地であって、前記培地が、請求項1〜10のいずれか一項に記載されたものである、培地。
- 胚を培養するためのin vitro方法であって、請求項17に記載の培地中で前記胚を培養する工程を含む方法。
- 前記胚が、ヒト胚である、請求項18に記載の方法。
- 胚の培養における、請求項17に記載の培地の使用。
- 前記培養が、基底膜と組み合わせて実施される、請求項11〜15又は17〜20のいずれか一項に記載の方法又は使用。
- 前記基底膜が、ラミニンを含む、請求項21に記載の方法又は使用。
- ヒト胚の培養に必須の因子についてスクリーニングする方法であって、
(i) 分析しようとする因子を添加することによって培地を調製する工程と;
(ii) 前記培地中でヒト胚を培養する工程と;
(iii) 前記胚を、前記因子を含まない培地中で又は前記因子の活性化剤若しくは阻害剤の存在下で培養した胚と比較する工程と
(iv) 前記因子が前記胚の培養に必須かどうかを決定する工程と
を含む方法。
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