JP2013046623A - 胚性幹細胞の誘導 - Google Patents
胚性幹細胞の誘導 Download PDFInfo
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- JP2013046623A JP2013046623A JP2012225193A JP2012225193A JP2013046623A JP 2013046623 A JP2013046623 A JP 2013046623A JP 2012225193 A JP2012225193 A JP 2012225193A JP 2012225193 A JP2012225193 A JP 2012225193A JP 2013046623 A JP2013046623 A JP 2013046623A
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Abstract
【解決手段】胚性幹(ES)細胞を作製する方法であって、胚から得られる割球を培養するステップであり、胚が生存し続けるステップを含む方法。所望の分化した細胞又は組織を作製する方法であって、胚から得られる割球を培養するステップであり、胚が生存し続けるステップと、割球の分化を誘発し所望の細胞又は組織を作製するステップとを含む方法。
【選択図】なし
Description
割球は、桑実胚のコンパクション前、桑実胚のコンパクション中、胞胚腔の形成前又は胚盤胞期中を含むがこれらに限定されない、着床に先立つ様々な発育段階において胚から摘出することができる。
・透明帯部分切開法(PZD):マイクロピペットを用いる透明帯の部分切開;
・透明帯開孔法:タイロード酸による部分消化を介する透明帯の化学的開口;
・透明帯開孔法:プロナーゼ又は他のプロテアーゼによる部分消化を介する透明帯の酵素的開口;
・透明帯菲薄化法:タイロード酸又はレーザーによる透明帯の菲薄化;
・レーザーによる透明帯の点状開口;
・ピエゾマイクロマニピュレーターによる透明帯の点状機械的開口が含まれる。
単離された割球は、すでに樹立された系からなどの胚性幹細胞、胚癌細胞、マウス胚線維芽細胞、他の胚様細胞、胚起源の細胞又は胚に由来する細胞が含まれるがこれらに限定されない任意の適当な細胞と一緒に標準培養条件で培地と一緒に培養器具上(例えば、マイクロウエル中)に置くことにより培養することができ、それらの多くは、当技術分野において知られており、the American Type Culture Collection、Manassas、VA 20110−2209、USA、及び他の供給源から入手可能である。これらの細胞は、割球の周りに集塊又は凝集する。マイクロウエルマイクロビーズを用いる方法若しくは懸滴法、又は当技術分野において知られているその他の凝集法を含む他の凝集法を使用することができる。特定の理論に束縛されたいわけではないが、数日又は数週間にわたり、培養された割球は、おそらく割球と共培養された胚細胞の間の細胞間相互作用の結果として、又は割球と胚細胞により分泌される因子の間の相互作用からES細胞成長を示すと考えられる。
また、本発明は、早期に胚盤胞前期胚をプレーティングして自己フィーダー細胞上に幹細胞を作製する方法を提供する。一実施形態において、この方法は、(a)胚盤胞前期胚を分割すること、(b)体細胞に直接分化させる条件下で組織培養液中に一方の部分をプレーティングし、フィーダー細胞を作製すること及び(c)胚盤胞前期胚の他の部分を自己フィーダー細胞上にプレーティングすることを含む。別の実施形態において、自己フィーダー細胞及びES細胞は、胚盤胞前期胚から摘出された割球から作製されるため、胚の着床される能力を保存する。
本発明の方法により作製されるES細胞の多能性は、ES細胞マーカータンパク質の発現を検出することにより決定することができる。そのようなタンパク質の例には、八量体結合タンパク質4(Oct−4)、発生段階特異的胚抗原(stage−specific emryonic antigen、SSEA)−1、Nanog、アルカリホスファターゼ及びRes−1が含まれるが、これらに限定されるものではない。一部の実施形態において、推定上のES細胞系は、13、20、30、40、50、60、70、80、90又は100を超える継代後に多能性を維持する。また、ES細胞を、正常な核型の維持についてアッセイすることができる。
また、本発明は、割球増殖体をFGF−4と接触させることにより、形態学的に栄養芽層及び/又は胚外内胚葉と似ているが、ES細胞と似ていない栄養芽層幹(「TS」)細胞を作製する方法を提供する。例えば、増殖体の培養培地にFGF−4を加える。TS細胞は、当技術分野において標準的な手順を用い、Nanog、Rex−1、及びCdx−2などのタンパク質の発現をアッセイすることにより検出することができる。また、TS細胞同定は、Oct−4及びα−フェトプロテインなどであるがこれらの限定されないタンパク質の発現がないことにより証明することができる。
本発明は、細胞治療に適している障害を治療する方法であって、治療有効量の本発明のES細胞を罹患対象に投与することを含む方法を提供する。本発明のES細胞は、ES細胞が有用であるいかなる使用にも適している。
実施例1:ES細胞系の作製
単一割球は、ピエゾパルスを用いて開けた透明帯の穴を介する生検か、或いはCa++/Mg++非含有PBS中で透明帯剥離胚を10分間脱凝集することのどちらかにより、8細胞期129/Sv−ROSA26:LacZマウス胚から単離した。生検された(7細胞)胚を、性交後日数(d.p.c.)1.5の同期化代理母の卵管に移し、各々の分離割球を、プラスチックの組織培養プレートの底に凝集針を押しつけることにより作成した300μmの凹み内で緑色蛍光タンパク質(GFP)陽性129Sv/CD−1マウスES(mES)細胞の小さな集塊(約100細胞)と一緒に凝集させた。24〜48時間のインキュベーション後に、GFP−mESクラスターの大部分の(60%)側面でGFP陰性細胞の増殖する「芽」が観察された(図4A、Bを参照)。細胞集塊を、マイトマイシンC処理マウス胚線維芽細胞(MEF)上にプレーティングし、ノックアウトDMEM(15%FCS、ペニシリン/ストレプトマイシン、Glutamax−I、β−メルカプトエタノール、非必須アミノ酸、LIF[2000U/ml]、及びMEK1阻害剤[50μM](mES培養培地))中で培養した。例えば、Hogan他「マウス胚の操作:実験室マニュアル(Manipulating the Mouse Embryos:A Laboratory Manual)」Cold Spring Harbor Laboratory Press;第2版、1994を参照されたい。割球の大部分(54/91)は、増殖する細胞集塊を4日以内に形成し、それらを、蛍光顕微鏡下でGFP陽性mES細胞から分離した。細胞を、機械的解離又はトリプシン処理により広げ、ES細胞と形態学的に似ているコロニーを選択し、すべてのGFP陽性細胞を排除した(図4、C、D、E、F)。
実施例2:ES細胞の分化
ES様細胞培養物を過剰増殖させた場合、筋肉アクチン(中胚葉)、βIIIチューブリン(外胚葉)、及びα−フェトプロテイン(原始内胚葉)に対する抗体による免疫染色から明らかなように、3つすべての胚層の細胞に自然発生的に分化した(図3B〜D及び図8A〜C)。拍動する心筋、胚外内胚葉及び複数の神経細胞タイプも、分化している培養液においてごく普通に観察された。誘導されたES細胞の多能性をさらに立証するため、ES細胞系を、CD−1マウス胚盤胞に注入するか、或いは前述のような(Hogan他、前掲書)8細胞期桑実胚と一緒に凝集させ、レシピエントの雌に移した。得られたキメラ胎児のX−Gal(5−ブロモ−4−クロロ−3−インドリル−β−ガラクトピラノシド)染色は、ES細胞系が、心臓、腎臓、肝臓、肺、腸、脳、血液、皮膚及び性隆起などのすべての器官に寄与していることを示した(図3A)。胎児のうちの24匹(83%)はキメラであり(図8F、G)、9匹の仔のうちの8匹(89%)はキメラであった(図8H)(後者は、配偶子中にLacZ遺伝子を有し(PCR分析により裏付けられた;図9)、CD−1と交配された場合にLacZ+子孫を生じ、生殖系列に対する割球由来ES細胞の寄与を裏付けた)。
実施例3:TS細胞系の作製
ES細胞ではなく栄養芽層及び胚外内胚葉と形態学的に似ている割球増殖体を、これらの条件下に維持されトリプシンと一緒に継代されたFGF−4産生栄養芽層幹(TS)様細胞50ng/mlと一緒にmES細胞培地中でさらに培養した。7つの推定上のTS系が樹立され、それらは、正常な核型を維持し、TS細胞のマーカーを発現した(図6B、D、F、H、J)。これらの細胞は、Oct−4(図6H)及びα−フェトプロテインについて陰性であった。推定上のTS細胞は、LacZ+TS細胞との凝集によって作製されるキメラ胎児中の胚外系列に寄与していた(図7)。RT−PCR分析は、これらの細胞が、Oct−4ではなくCdx−2を発現することを裏付けた(図5C及びF)。Nanog及びRex−1は、推定上のTS細胞系とES細胞系の双方で発現された(図5D及びE)。
Claims (48)
- 胚性幹(ES)細胞を作製する方法であって、胚から得られる割球を培養するステップであり、胚が生存し続けるステップを含む方法。
- 割球が、桑実胚のコンパクションに先立って胚から得られる請求項1に記載の方法。
- 割球が、胞胚腔の形成前に胚から得られる請求項1に記載の方法。
- 割球が、胚を取り囲む透明帯を部分的又は完全に除去することにより得られる請求項1に記載の方法。
- 割球が、胚由来細胞と一緒に培養される請求項1に記載の方法。
- 胚由来細胞が、ES細胞又は胚癌細胞である請求項5に記載の方法。
- 割球が、ES細胞の分化を阻害する因子と一緒に培養される請求項1に記載の方法。
- 割球を培養してES細胞を作製するステップ中に、組換えOct−4が割球中に導入されるか、又は内因性Oct−4が割球内で活性化される請求項1に記載の方法。
- ES細胞が、多能性又は全能性である請求項1に記載の方法。
- ES細胞が、ES細胞マーカータンパク質を発現する請求項1に記載の方法。
- ES細胞マーカータンパク質が、Oct−4、SSEA−1、Nanog、アルカリホスファターゼ又はRes−1である請求項10に記載の方法。
- ES細胞が、哺乳類細胞である請求項1に記載の方法。
- 割球が、細胞分裂を起こし、1つの子孫細胞が、遺伝子検査に使用され、異なる子孫細胞が、ES細胞を作製するのに使用される請求項1に記載の方法。
- 割球が、自己フィーダー細胞と一緒に培養され、フィーダー細胞が、割球を体細胞に分化させる条件下で胚から得られる割球を培養してフィーダー細胞を作製することにより得られる請求項1に記載の方法。
- ES細胞を作製する方法であって、
(a)胚から割球を得るステップであり、胚が生存し続けるステップと、
(b)割球をES細胞と一緒に凝集させるステップと、
(c)凝集した割球及びES細胞を割球がES細胞の特性を示すまで培養するステップと、
(d)割球に由来するES細胞を単離するステップと
を含む方法。 - 請求項1に記載の方法により作製されるES細胞。
- ES細胞が、全能性又は多能性である請求項16に記載のES細胞。
- 細胞が、ES細胞マーカータンパク質を発現する請求項16に記載のES細胞。
- ES細胞マーカータンパク質が、Oct−4、SSEA−1、Nanog、アルカリホスファターゼ又はRes−1である請求項18に記載のES細胞。
- ES細胞が、哺乳類細胞である請求項16に記載のES細胞。
- 請求項16に記載のES細胞に由来する分化した細胞又は組織。
- 細胞又は組織が、中胚葉性、内胚葉性又は外胚葉性である請求項21に記載の分化した細胞又は組織。
- ES細胞系を作製する方法であって、請求項20に記載のES細胞を培養してES細胞系を作製するステップを含む方法。
- 請求項23に記載の方法により作製されるES細胞系。
- 所望の分化した細胞又は組織を作製する方法であって、請求項16に記載のES細胞の所望の細胞又は組織への分化を誘発するステップを含む方法。
- 所望の分化した細胞又は組織を作製する方法であって、胚から得られる割球を培養するステップであり、胚が生存し続けるステップと、割球の分化を誘発し所望の細胞又は組織を作製するステップとを含む方法。
- 請求項25又は26に記載の方法により作製される分化した細胞又は組織。
- 組織又は細胞が、中胚葉性、内胚葉性又は外胚葉性である請求項27に記載の分化した細胞又は組織。
- 患者において細胞治療に適している障害を治療する方法であって、請求項16に記載のES細胞又は請求項21若しくは27に記載の分化した細胞若しくは組織を患者に投与するステップを含む方法。
- 請求項16に記載のES細胞又は請求項21若しくは27に記載の分化した細胞若しくは組織、及び薬学的に許容できる媒体又は担体を含む医薬組成物。
- 栄養芽層幹(TS)細胞を作製する方法であって、胚から得られる割球を培養するステップであり、胚が生存し続けるステップを含む方法。
- 割球が、桑実胚のコンパクションに先立って胚から得られる請求項31に記載の方法。
- 割球が、胞胚腔の形成前に胚から得られる請求項31に記載の方法。
- 割球が、胚を取り囲む透明帯を部分的又は完全に除去することにより得られる請求項31に記載の方法。
- 割球が、胚由来細胞と一緒に培養される請求項31に記載の方法。
- 割球が、FGF−4と一緒に培養される請求項31に記載の方法。
- TS細胞が、TS細胞マーカータンパク質を発現する請求項31に記載の方法。
- TS細胞マーカータンパク質が、Nanog、Rex−1又はcdx−2である請求項31に記載の方法。
- TS細胞が、Oct−4又はα−フェトプロテインを発現しない請求項31に記載の方法。
- TS細胞を作製する方法であって、
(a)胚から割球を得るステップであり、胚が生存し続けるステップと、
(b)割球をES細胞と一緒に凝集させるステップと、
(c)割球から増殖体を得るステップであり、増殖体が栄養芽層又は胚外内胚葉細胞の特性を示すステップと
(d)増殖体をFGF−4と接触させてTS細胞を作製するステップと、
(e)割球に由来するTS細胞を単離するステップとを含む方法。 - TS細胞が、哺乳類細胞である請求項31に記載の方法。
- 請求項31に記載の方法により作製されるTS細胞。
- TS細胞が、TS細胞マーカータンパク質を発現する請求項42に記載のTS細胞。
- TS細胞マーカータンパク質が、Nanog、Rex−1又はcdx−2である請求項43に記載のTS細胞。
- TS細胞が、哺乳類細胞である請求項42に記載のTS細胞。
- 請求項42に記載のTS細胞に由来する分化した細胞又は組織。
- TS細胞系を作製する方法であって、請求項42に記載のTS細胞を培養してTS細胞系を作製するステップを含む方法。
- 請求項47に記載の方法により作製されるTS細胞系。
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