JP2005013152A - Cell differentiation retarder and method for culturing cell, culture solution and cultured cell by using the same retarder - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell differentiation retarder obtained by culturing a stem cell or a embryonic stem cell in a undifferentiated state without using a feeder cell and to provide a culturing method using the cell differentiation retarder and to provide a culture solution by using the culturing method and a cell cultured and prepared by using the cell differentiation retarder. <P>SOLUTION: Enhancing effect of undifferentiated and pluripotent embryonic stem cell is found in a low molecular compound, particularly indole derivative. The stem cell differentiation retarder comprises the low molecular compound, particularly the indole derivative as an active ingredient. The method for culturing the embryonic stem cell comprises culturing the embryonic stem cell in large amount in safe and undifferentiated state in the absence of the feeder cell by using the indole derivative. The culture solution of the stem cell comprises the indole derivative. The cell is cultured and prepared by using the indole derivative as the cell differentiation retarder. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００１１】
（式中、Ｒ^１〜Ｒ^９は、同一または異なった電子吸引基、電子供与基または水素電子を示す。具体的には、アルキル基、アルコキシ基、ヒドロキシル基、ニトロ基、アリールアミノビニル基、水素原子などが例示される）。
【００１２】
さらに、本発明は、前記の幹細胞分化抑制剤を用いて幹細胞、特に胚性幹細胞を未分化状態で培養する方法に関する。
また、本発明は、前記の幹細胞分化抑制剤を用いた幹細胞の培養液に関する。
さらに、本発明は、前記の幹細胞分化抑制剤を用いて培養し作製された細胞に関する。
【００１３】
本発明によれば、幹細胞、好ましくは胚性幹細胞を、未分化なまま、期間を延長してまたは無期限に、大量にかつ安全に増殖させることができる。本発明によって提供される分化抑制剤、それを用いた培養方法、培養液は、細胞移植用途で用いる細胞ソースとしての幹細胞、好ましくは胚性幹細胞を産生するために適用され得る。幹細胞、好ましくは胚性幹細胞を利用することにより得られる多くの恩恵に加え、本発明によって提供される分化抑制剤およびそれを用いた培養方法は、１つもしくは多数の遺伝的な改変を有する胚性幹細胞を産生するために適用され得る。このような適用の例は、疾患について細胞ベースでのモデルの開発、ならびに遺伝病を処置するために移植について特異化された組織の開発を含むが、これに限定されない。
【００１４】
以下の用語は他で述べない限り、この節中で提供されるように定義される。本明細書で使用される他の全ての用語は、他に述べない限り、その用語に関する特定の分野でのその語法に関して定義される。
幹細胞：幹細胞とは、特異化された機能を有する他の細胞型、即ち、最終的に分化した細胞、もしくは、より狭い範囲の細胞型に分化可能な他の幹細胞型に分化し得る細胞を指す。
全能性幹細胞：全能性幹細胞とは、多能性の細胞および完全に分化した細胞（すなわち、種々の細胞へと、もはや分化し得ない細胞）を含む任意の細胞型へと分化し得る細胞のことを言う。
多能性幹細胞：多能性幹細胞とは、必ずしも全ての型にならないけれども、異なる多数の細胞型のうちの１つへと分化し得る細胞をいう。多能性細胞の１つの例は、骨髄幹細胞であり、この細胞は神経細胞以外の、リンパ球および赤血球のような種々の血液細胞型へと分化し得る。従って、全ての全能性細胞は多能性であるのに対して、全ての多能性細胞が全能性であるわけではないことが認識される。
胚性幹細胞：胚性幹細胞とは、幹細胞の中でも特に前着床段階の胚の、桑実胚または胚盤胞段階から得られた全能性の細胞をいい、ＥＳ細胞とも呼ばれる。 また、胚性幹細胞には、精子あるいは卵子になると決まっている、胚または胎児（胎仔）の始原生殖細胞に由来する多能性の幹細胞のことをいう場合もある。ただし、この細胞は胚性生殖（Ｅｍｂｒｙｏｎｉｃ Ｇｅｒｍ、ＥＧ）細胞と呼ばれて胚性幹細胞と区別される場合もある。本明細書中で用いる胚性幹細胞は、いかなる動物種のものであってよく、例えばヒトを含む霊長類、霊長類以外の哺乳類、鳥類などの胚性幹細胞が挙げられる。
全能性：全能性とは、多能性の細胞および完全に分化した細胞（すなわち、種々の細胞へと、もはや分化し得ない細胞）を含む任意の細胞型へと分化し得る状態を言う。
多能性：多能性とは、必ずしも全ての型にならないけれども、異なる多数の細胞型のうちの１つへと分化し得る状態をいう。
未分化：未分化とは、１つの細胞、或いは複数の細胞からなる任意の細胞集団が、１つまたは複数の、さらに分化が進んだ状態の細胞に分化し得る能力を有する状態である細胞、或いは該細胞を含む細胞集団である状態であることをいう。
フィーダー：本発明を記載する目的のために用いられるフィーダーとは、 全能性幹細胞がその上にプレートされ、プレートされた全能性幹細胞の増殖の助けとなる環境を提供するものをいう。
フィーダー細胞：本発明を記載する目的のために用いられるフィーダー細胞とは、 全能性幹細胞がその上にプレートされる非全能性幹細胞をいい、非全能性幹細胞は、プレートされた全能性幹細胞の増殖の助けとなる環境を提供する。
細胞由来成分：細胞から分泌される成分、内容物、および細胞膜成分など、細胞に由来する全ての成分をいう。
【００１５】
本発明は、幹細胞、好ましくは胚性幹細胞を未分化の状態で、増殖および維持させ得る分化抑制剤、それを用いた培養方法、それを用いた培養液、それを用いて培養し作製された細胞株を提供する。本発明で提供する分化抑制剤、培養方法および培養液は、従来より簡便に、安全に、未分化の胚性幹細胞を増殖しそして維持する。本発明の分化抑制剤を含む細胞培養方法はまた、特定の分化誘導因子、および分化誘導因子の有用な組み合わせについてスクリーニングするために使用され得る。本発明の分化抑制剤、および培養方法を使用して、未分化な胚性幹細胞を増殖させる能力は、重要な治療適用を有する単一もしくは複数の遺伝的改変を有する胚性幹細胞系を産生する能力を含む重要な利益を提供する。
【００１６】
本発明の分化抑制剤は、化学的に安定な低分子化合物で、幹細胞を未分化な状態で維持する活性を有するものであればいずれも用いることができるが、好ましくはインドール誘導体があげられる。さらに好ましくは式（Ｉ）で示される構造を有するインドール誘導体、その塩、またはエステル化合物が望ましい。式中、Ｒ^１〜Ｒ^９としては、同一または異なった電子吸引基、または電子供与基または水素原子から選ばれる基、または原子が挙げられる。電子供与基とは、ベンゼン環へ電子を供与し得る置換基、電子吸引基とはベンゼン環上のπ電子を吸引する性質を有する置換基をいう。また、Ｈａｍｍｅｔｔの置換基定数σを用いてσ＜０を電子供与基、σ＞０を電子吸引基と定義することもできる（基礎有機反応論、橋本静信ら著、三共出版、１９９７年）。より好ましくは、 Ｒ^１〜Ｒ^９は、同一または異なった、アルキル基、アルケニル基、アルキニル基、フェニル基、ナフチル基、フリル基、チエニル基、アルコキシ基、アルキルアミノ基、アルキルカルボニル基、ベンゾイル基、ナフトイル基、フロイル基、テノイル基、ジアルキルカルバモイル基、アセチル基、ブタノイル基、メトキシカルボニル基、シクロアルキル基、ベンジルオキシ基、アダマンチルオキシ基、アリールアミノビニル基、ヒドロキシル基、ニトロ基よりなる群から選ばれる基、あるいはハロゲン原子または水素原子などが挙げられる。さらには、 Ｒ^１〜Ｒ^９はアルキル基、アルコキシ基、ヒドロキシル基、ニトロ基、アリールアミノビニル基、水素原子よりなる群から選ばれる基または原子であることが望ましい。
【００１７】
本発明はこれらの化合物のうち、次の式（ＩＩ）で示されるインドール誘導体またはその塩を用いることが特に好ましい。
【化４】

Ａ^１〜Ａ^３は同一であっても良い低級アルキル基、低級アルコキシ基、水素原子を表し、Ａ^４は低級アルキル基、アリールアミノビニル基を表す。
【００１８】
化合物（ＩＩ）において、Ａ^１〜Ａ^４で示される低級アルキル基としては、例えばメチル、エチル、プロピル、イソプロピル、ブチル、イソブチル、ｓｅｃ−ブチル、ｔｅｒｔ−ブチル、ペンチル、ヘキシルなどが挙げられる。特に好ましくはメチルである。
【００１９】
化合物（ＩＩ）において、Ａ^１〜Ａ^４で示される低級アルコキシ基としては、例えばメトキシ、エトキシ、プロポキシ、イソプロポキシ、ブトキシ、イソブトキシ、ｓｅｃ−ブトキシ、ｔｅｒｔ−ブトキシ、ペンチルオキシ、ヘキシルオキシなどが挙げられるが、特に好ましくはメトキシである。
【００２０】
化合物（ＩＩ）において、Ａ^４で示されるアリールアミノビニル基としては式（ＩＩＩ）で示される基を用いることが望ましい。式（ＩＩＩ）で示されるＡｒ（アリール基）としては、例えば、フェニル基、トリル基、メトキシフェニル基、キシリル基、ナフチル基、アントリル基などが挙げられるが、特に好ましくは、４−メトキシフェニル基である。
【化５】

【００２１】
化合物（ＩＩ）の塩としては、薬学的に許容しうる塩が望ましい。例えば、塩酸塩、硫酸塩、リン酸塩、臭化水素酸塩、酢酸塩、マレイン酸塩、フマル酸塩、コハク酸塩、メタンスルホン酸塩、ｐ−トルエンスルホン酸塩、クエン酸塩、酒石酸塩などを形成しても良い。
【００２２】
化合物ＩまたはＩＩのエステル化合物としては、カルボン酸エステル、スルホン酸エステル、無機酸エステルなどがあげられる。
【００２３】
本発明の分化抑制剤の濃度としては、０．１ｎｇ／ｍｌ〜１ｍｇ／ｍｌの範囲で使用することが望ましく、好ましくは１０ｎｇ／ｍｌ〜１００μｇ／ｍｌ、さらに好ましくは１００ｎｇ／ｍｌ〜１０μｇ／ｍｌの範囲で使用することである。
【００２４】
また、本発明の分化抑制剤はＳＴＡＴ３（ｓｉｇｎａｌ ｔｒａｎｓｄｕｃｅｒ ａｎｄ ａｃｔｉｖａｔｏｒ ｏｆ ｔｒａｎｓｃｒｉｐｔｉｏｎ ３）の活性化を介さずに胚性幹細胞の未分化を維持する低分子化合物、特にイソキノリン誘導体を含む。特定のマウス系統の胚性幹細胞は、マウス胎仔由来の線維芽細胞からなるフィーダー細胞の存在、非存在にかかわらず、ＬＩＦによって未分化が維持される。ＬＩＦはＳＴＡＴ３の活性化を介して下流にシグナルを伝えることが知られている（Ｍａｔｓｕｄａら、ＥＭＢＯ Ｊｏｕｒｎａｌ、１８、１５、ｐ４２６１、１９９９年）。しかし、最近になってＳＴＡＴ３を活性化せずに胚性幹細胞の未分化を維持する分化抑制因子が存在することが報告されたことから（Ｄａｎｉら、Ｄｅｖｅｌｏｐｍｅｎｔａｌ Ｂｉｏｌｏｇｙ、 ２０３、 ｐ１４９、 １９９８年）、ＳＴＡＴ３を介さない胚性幹細胞の未分化維持機構が存在すると考えられている。さらに、ＬＩＦが、霊長類胚性幹細胞の未分化維持に効果を示さないことから（Ｔｈｏｍｓｏｎら、Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ， ９２， ｐ７８４４， １９９５年、Ｔｈｏｍｓｏｎら、Ｓｃｉｅｎｃｅ， ２８２， ｐ１１４５， １９９８年、Ｒｅｕｂｉｎｏｆｆら、 Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈ， １８， ｐ３９９， ２０００年）、霊長類胚性幹細胞には、ＬＩＦおよびＳＴＡＴ３とは異なるシグナルによって未分化状態を維持する機構が存在することが示唆される。本発明の分化抑制剤にはＳＴＡＴ３の活性化を介さずに胚性幹細胞の未分化を維持する低分子化合物が含まれる。いいかえれば、ＬＩＦとは異なる作用により胚性幹細胞の未分化を維持する活性を有する低分子化合物が含まれる。
【００２５】
また、最近の報告によれば、ある種の骨髄由来間葉系幹細胞においても胚性幹細胞と同様に、マウス由来の幹細胞の培養はＬＩＦ依存的であるが、ヒト由来の幹細胞はＬＩＦ非依存的であることが示されている。（Ｖｅｒｆａｉｌｌｉｅら、Ｎａｔｕｒｅ， ４１８， ｐ４１， ２００２年）これは、多分化能を示す幹細胞は、胚性幹細胞と同様の未分化維持機構を有していることを示唆し、従って、霊長類幹細胞においても、胚性幹細胞と同様に、ＬＩＦ−ＳＴＡＴ３経路とは異なるシグナルによって未分化が維持されている可能性が示唆される。本発明の分化抑制剤には、ＳＴＡＴ３を介さずに細胞の未分化状態を維持する活性を有する低分子化合物が含まれる。
【００２６】
本発明の分化抑制剤は、動物細胞培養用基礎培地である任意の哺乳類細胞培養基本培地に添加して使用することができる。動物細胞基本培地の例としては、ダルベッコ改変イーグル培地：ＤＭＥＭ、ノックアウトＤＭＥＭ、グラスゴーＭＥＭ：ＧＭＥＭ、ＲＰＭＩ１６４０、ＩＭＤＭ（以上 ＧＩＢＣＯ社製、米国）などが挙げられるがこれらに限定されない。１つの実施態様としての細胞培地はダルベッコ改変イーグル培地（ＤＭＥＭ）である。さらにこれらの基礎培地に血清または血清代替物としての各種増殖因子を添加して用いることもできる。血清は、幹細胞ならびに胚性幹細胞の増殖および生存性の維持に効果的である栄養素を供給する任意の血清、または、血清ベースの溶液であり得る。このような血清の例には、ウシ胎仔血清（ＦＣＳ）、ウシ血清（ＣＳ）、馬血清（ＨＳ）などがあり、また、血清代替物としては当業者に周知のもの、蛋白質、アミノ酸、脂質、ビタミンなどを単独で、或いは組み合わせて用いることが出来る。蛋白質としてはインスリン、トランスフェリン、アルブミン、ペプトン、ＦＧＦ（Ｆｉｂｒｏｂｌａｓｔ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ）、ＥＧＦ（ＥｐｉｔｈｅｒｉａｌＧｒｏｗｔｈ Ｆａｃｔｏｒ）などが、アミノ酸としてはアルギニン、システイン、グルタミン、グリシン、ヒスチジン、イソロイシン、ロイシン、リジン、メチオニン、フェニルアラニン、セリン、スレオニン、トリプトファン、チロシン、バリンなどが、ビタミンとしては、パントテン酸、コリン、葉酸、イノシトール、ニコチン酸アミド、リボフラビン、チアミン、ピリドキシンなどが例示されるがこれらに限定されない。１つの実施態様において、血清はウシ胎仔血清である。より特定の実施態様において、ウシ胎仔血清は約２５％と約１％との間の濃度で提供される。さらにより特定の実施態様において、細胞培地でのウシ胎仔血清濃度は１５％である。また、他の実施態様において、血清代替物はノックアウト血清リプレースメント：ＫＳＲ（ＧＩＢＣＯＢＲＬ社製、米国）である。
【００２７】
細胞培地は、抗酸化剤（還元剤）（例えば、β−＜メルカプトエタノール）も含む。ある好適な実施態様において、β−＜メルカプトエタノールは、約０．１ｍＭの濃度を有する。他の抗酸化剤 （例えば、モノチオグリセロール、もしくは、ジチオス レイトール（ＤＴＴ）の単独もしくは組み合わせ）が同様の効果のために使用され得る。さらに他の等価な物質は、細胞培養の分野の当業者に周知である。
【００２８】
本発明の分化抑制剤ならびにその有効成分は、任意の培養基材とともにあるいは培養基材に固定して使用することもできる。培養基材には多孔質体を使用することがでる。多孔質体とは、微細な孔を多数有する基材のことをいい、その材質、厚さ、形状、寸法などは特に限定はない。多孔質体の材質は、有機材料、無機材料及び有機材料と無機材料からなる複合材料であっても良い。多孔質体の形状は平板状、球状、棒状、繊維状、中空状のいずれの形態であっても良く、例えば、フィルム、シート、膜、板、不織布、ろ紙、スポンジ、織物、編物、塊、糸、中空糸、粒子等が挙げられる。細胞を培養するにあたって、３次元的に培養できるように細胞を支持する孔の大きさを簡単に制御できることや、基材作製の容易さ及びコストなどを考慮すると不織布がより好ましい。多孔質体の孔の大きさについては、特に限定はないが、細胞を３次元的に支持できるようにすることを考慮すると、平均孔径が、０．１μｍ以上１００μｍ以下であることが好ましく、１μｍ以上５０μｍ以下がさらに好ましい。繊維径については特に限定はないが、０．０３デニール以下が好ましい。
【００２９】
上記の多孔質体は、細胞の接着性や分化維持機能、増殖能を向上させるために、高分子化合物により表面コーティング処理を施されていても良い。高分子物質とは、１種以上の繰り返し構成単位の単量体が１次元、２次元、３次元的に連なった分子量数百以上の物質のことをいう。高分子物質は、大きく天然高分子物質、半合成高分子物質、合成高分子物質の３つに分類することができ、本発明においていずれの高分子物質も使用することができる。
【００３０】
例えば、天然高分子物質としては、マイカ（雲母）、アスベスト（石綿）、グラファイト（石墨）、ダイアモンド、でんぷん、セルロース、アルギン酸等に代表される糖類及びゼラチン、フィブロネクチン、フィブリノーゲン等に代表されるタンパク質等が挙げられる。半合成高分子物質としては、ガラス、硝酸セルロース、酢酸セルロース、塩酸ゴム、カルボキシメチルセルロース等が挙げられる。合成高分子物質としては、ポリホスホニトリルクロライド、ポリエチレン、ポリ塩化ビニル、ポリアミド、ポリエチレンテレフタレート、ポリスルホン、ポリアクリロニトリル、ポリビニルアルコール、ポリメチルメタクリレート、ポリヒドロキシエチルメタクリレート、ポリジメチルアミノエチルメタクリレート及びヒドロキシエチルメタクリレートとジメチルアミノエチルメタクリレートの共重合体に代表されるような２種類以上の合成単量体からなる共重合体等が挙げられる。
コーティング処理のし易さを考慮すると、有機高分子物質が好ましく、タンパク質やペプチド並びに有機合成高分子物質がさらに好ましい。
【００３１】
本発明の分化抑制剤を固定した培養基材を細胞捕捉材として用いることにより、複数の異なる細胞からなる集団から、幹細胞或いは胚性幹細胞を分離し、かつ、培養を行える方法及び装置が提供され得る。即ち、幹細胞或いは胚性幹細胞と、除去対象細胞を含む細胞含有液を本発明の分化抑制剤を固定した、多孔質体などの培養基材からなる細胞捕捉材が充填されている容器に導入し、細胞捕捉材に幹細胞或いは胚性幹細胞を捕捉させ、除去対象細胞を容器外に導出した後に容器ごと培養することを特徴とする幹細胞或いは胚性幹細胞培養方法であり、また本発明の分化抑制剤を固定化した培養基材からなる細胞捕捉材を容器に充填した細胞培養装置であって、前記細胞捕捉材は細胞培養用担体として使用し得るものであり、前記容器は細胞培養に使用し得るものであることを特徴とする細胞培養装置である。除去対象細胞とは、幹細胞或いは胚性幹細胞以外の全ての細胞をいう。また、幹細胞或いは胚性幹細胞から分化して、多分化能を失った細胞もこれに含まれる。細胞捕捉材に導入する細胞含有液としては、幹細胞或いは胚性幹細胞を含有する細胞液であればいかなるものでもよく、一例として、血液、骨髄、砕片組織液、或いは、幹細胞、胚性幹細胞の培養液などがあげられる。
【００３２】
本発明は幹細胞ならびに胚性幹細胞を増殖するための方法に関する。本発明の分化抑制剤を用いて増殖させた幹細胞ならびに胚性幹細胞の培養物を提供することができる。
【００３３】
本発明の分化抑制剤を用いて培養される細胞には、公知の方法および材料を使用して入手し得る全ての幹細胞および胚性幹細胞が含まれる。幹細胞には以下の公知の方法によって入手し得る幹細胞が一例として挙げられる。骨髄細胞（「骨髄移植ガイド」Ｈ．Ｊ．ディーグ、Ｈ．Ｇ．クリンゲマン、Ｇ．Ｌ．フィリップス著／笠倉新平訳）、骨髄幹細胞（Ｏｓａｗａら、 Ｓｃｉｅｎｃｅ、 ２７３、 ｐ２４２−２４５、１９９６年、 Ｇｏｏｄｅｌｌら、 Ｊ．Ｅ．Ｍｅｄ．１８３、 ｐ１７９７−１８０６、１９９６年、Ｖｅｒｆａｉｌｌｉｅら、Ｎａｔｕｒｅ， ４１８， ｐ４１， ２００２年）、神経幹細胞（Ｒｅｙｎｏｌｄｓら、Ｓｃｉｅｎｃｅ、ｐ１７０７−１７１０、１９９２年）、組織幹細胞（Ｇｏｏｄｅｌｌら、Ｊ．Ｅ．Ｍｅｄ．１８３、ｐ１７９７−１８０６、１９９６年、松崎ら、実験医学、１９、ｐ３４５−３４９、２００１年、Ｂｌａｕら、Ｃｅｌｌ、１０５、ｐ８２９−８４１、２００１年）、間葉系幹細胞（Ｌｉｅｃｈｔｙら、Ｎａｔｕｒｅ Ｍｅｄｉｃｉｎｅ、６、ｐ１２８２−１２８６、２０００年、Ｐｉｔｔｅｎｇｅｒら、Ｓｃｉｅｎｃｅ、２８４、ｐ１４３−１４７、１９９９年）。
【００３４】
また、培養されるべき胚性幹細胞としては、 以下に示す、公知の方法および材料を使用して入手し得る。マウス胚性幹細胞：Ｅｖａｎｓら、Ｎａｔｕｒｅ， ２９２， ｐ１５４， １９８１年、ウシＥＳ細胞：Ｓｃｈｅｌｌａｎｄｅｒら、Ｔｈｅｒｉｏｇｅｎｏｌｏｇｙ，３１，ｐ１５−１７，１９８９年、豚ＥＳ細胞：Ｓｔｒｏｊｅｋら、Ｔｈｅｒｉｏｇｅｎｏｌｏｇｙ，３３：ｐ９０１， １９９０年、羊ＥＳ細胞：Ｈａｎｄｙｓｉｄｅ、Ｒｏｕｘ’ｓ Ａｒｃｈ．Ｄｅ ｖ．Ｂｉｏｌ．，１９６：ｐ１８５，１９８７年、ハムスターＥＳ細胞：Ｄｏｅｔｓｃｈｍａｎら，Ｄｅｖ．Ｂｉｏｌ．，１２７、ｐ２２４，１９８８年、 サルＥＳ細胞：Ｔｈｏｍｓｏｎら、Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ， ９２， ｐ７８４４， １９９５年、ヒトＥＳ細胞：Ｔｈｏｍｓｏｎら、Ｓｃｉｅｎｃｅ， ２８２， ｐ１１４５， １９９８年、Ｒｅｕｂｉｎｏｆｆら、Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈ， １８， ｐ３９９， ２０００年、ヒトＥＧ細胞：Ｇｅａｒｈａｒｔら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ， ９５， ｐ１３７２６， １９９８年。また、マウス胚性幹細胞（１２９ＳＶおよびＣ５７／ＢＬ６）は、大日本製薬より購入できる。
【００３５】
本発明により提供される分化抑制剤は、全ての幹細胞および胚性幹細胞に用いることができるが、哺乳類の幹細胞および胚性幹細胞に用いることが望ましく、霊長類の幹細胞および胚性幹細胞に用いることが好ましい。
【００３６】
幹細胞ならびに胚性幹細胞は、一旦単離されると、本発明の分化抑制剤を使用して未分化な状態で培養され得る。
【００３７】
本発明の分化抑制剤を用いて培養した幹細胞、好ましくは胚性幹細胞の未分化程度は幹細胞の細胞膜状に存在するアルカリホスファターゼ（ＡＬＰ）活性を測定することにより確認することができる。未分化な胚性幹細胞ではＡＬＰの活性が維持され、分化すると減少することが知られている（Ｗｉｌｌｉａｍｓら、Ｎａｔｕｒｅ、３３６、ｐｐ６８４、１９８８年、Ｔｈｏｍｓｏｎら、Ｓｃｉｅｎｃｅ， ２８２， ｐ１１４５， １９９８年）。不溶性基質を用いた染色法、あるいは水溶性基質を用いた分光学的測定法などによりアルカリホスファターゼ（ＡＬＰ）活性を検出する方法が挙げられる。
【００３８】
一つの実施態様において、分光学的測定法によりＡＬＰ活性を定量することができる。培養ディッシュ上の細胞にパラニトロフェニルホスフェイト（ｐＮＰＰ）のアルカリ性溶液を添加する。細胞膜上に存在するＡＬＰによってｐＮＰＰが加水分解され、パラニトロフェノールが生じる。生成した溶液の４０５ｎｍの吸光度を測定することによって、アルカリホスファターゼ活性を定量することができる。実施例４に記載の方法により、本発明の分化抑制剤を添加して培養した胚性幹細胞のＡＬＰ活性を測定すると、分化抑制剤を含まない培地にて培養したコントロールの胚性幹細胞にくらべ有意に高いＡＬＰ活性を有していることが示される。即ち本発明の分化抑制剤が、胚性幹細胞の未分化状態を維持して増殖させ得ることが確認される。
【００３９】
他の実施態様において、ＡＬＰ染色法によりＡＬＰ活性を検出することもできる。培養ディッシュ上の細胞に、基質としてリン酸エステル塩とジアゾニウム塩を含む反応溶液を添加する。細胞膜状に存在するアルカリホスファターゼによりリン酸エステル塩が加水分解され、次いでジアゾニウム塩とカップリング反応することによりアゾ色素が生じ、ＡＬＰ活性部位に色素が沈殿する。染色されたコロニー数を計測することにより細胞のＡＬＰ活性を定量化することが可能となり、即ち細胞の未分化度合いを定量することができる。本発明の分化抑制剤を用いて培養した胚性幹細胞をＡＬＰ染色すると、分化抑制剤を用いないコントロール細胞に比べ有意にＡＬＰ活性が高いことが示され、即ち本発明の分化抑制剤が、胚性幹細胞の未分化状態を維持して増殖させ得ることが確認され
る。
【００４０】
また、胚性幹細胞の未分化程度はＯｃｔ−３／４遺伝子の発現量を測定することによって確認することができる。Ｏｃｔ−３／４遺伝子はＰＯＵファミリーに属する転写因子で、胚性幹細胞、胚性癌細胞（ＥＣ細胞）で、未分化状態特異的に発現し（Ｏｋａｍｏｔｏら、Ｃｅｌｌ、６０、ｐ４６１、１９９０年）、胚発生においても未分化細胞系譜においてのみ発現し（Ｓｃｈｏｌｅｒ、Ｔｒｅｎｄｓ Ｇｅｎｅｔ、７、 ｐ３２３、 １９９１年）、さらに、Ｏｃｔ−３／４遺伝子破壊マウスのホモ個体は胚盤胞期で発生を停止することから未分化状態維持に重要な機能を有していることが明らかにされている（Ｎｉｃｈｏｌｓら、Ｃｅｌｌ、 ９５、 ｐ３７９、 １９９８年）。また、一方、Ｏｃｔ−３／４遺伝子の過剰発現は胚性幹細胞の分化を促進することが最近明らかにされ（Ｎｉｗａら、Ｎａｔ ．Ｇｅｎｅｔ． ２４、 ｐ３７２、２０００年）、Ｏｃｔ−３／４の発現量をある一定の範囲に保つことが未分化状態の維持に重要である。Ｏｃｔ−３／４遺伝子の発現量を測定する一つの実施態様としては定量的ＰＣＲ（ポリメラーゼ連鎖反応）法を用いることができる。
【００４１】
一つの実施態様においてはリアルタイムＰＣＲ法が用いられ、幅広いダイナミックレンジをもち、簡便で信頼性のある定量測定が可能である。リアルタイムＰＣＲ技術には、ＡＢＩ ＰＲＩＳＭ７７００ＴＭ （Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を使用したＴａｑ Ｍａｎプローブを用いる方法や、ＬｉｇｈｔＣｙｃｌｅｒＴＭ（ロシュ・ダイアグノスティック）を用いた方法がある。特に後者の場合はＰＣＲの温度サイクルが数十分で終了する高速反応サイクルのもとで、サイクルごとに合成されるＤＮＡの増幅量変化をリアルタイムに検出できる。リアルタイムＰＣＲ法のＤＮＡ検出法としては、ＤＮＡ結合色素（インターカレーター）、ハイブリダイゼーション・プローブ（キッシングプローブ）、ＴａｑＭａｎプローブおよびＳｕｎｒｉｓｅユニプライマー（モレキュラー・ビーコン）を利用する４種類の方法がある。また、ＤＮＡ結合色素、例えばＳＹＢＲ ＧｒｅｅｎＩを利用してＯｃｔ−３／４遺伝子の発現量を解析することができる。ＳＹＢＲ ＧｒｅｅｎＩはＤＮＡの二本鎖特異的に結合色素であり、二本鎖に結合することで本来の蛍光強度が増強される。ＰＣＲ反応時にＳＹＢＲ ＧｒｅｅｎＩを加え、伸張反応の各サイクルの終わりに蛍光強度を測定すれば、ＰＣＲ産物の増加が検出できる。Ｏｃｔ−３／４遺伝子を検出するには通常のＰＣＲと同様にＯｃｔ−３／４遺伝子の配列をもとに、市販の遺伝子解析ソフトウェアなどを用いてプライマーを設計する。ＳＹＢＲ ＧｒｅｅｎＩは非特異的産物も検出してしまうため最適なプライマーの設定が必要となる。設計基準としては、オリゴマーの長さ、配列の塩基組成、ＧＣ含量、およびＴｍ値などに留意が必要である。
【００４２】
多くの場合、定量ＰＣＲにおいて明らかにすることを目的とするのは、サンプル一定量当たりの目的ＤＮＡ量である。このためには最初に反応系に加えたサンプル量の評価が必要である。この場合サンプル量を反映するような内部標準となる別のＤＮＡを目的ＤＮＡとは別に測定し、最初に反応系に加えたサンプル量を補正することができる。サンプル量を補正する目的で用いる内部標準には、通常、組織によって発現量に差がないと考えられているハウスキーピング遺伝子を用いることができる。例えば、解糖系の主要酵素であるグリセロアルデヒドリン酸脱水素酵素（ＧＡＰＤＨ）、細胞骨格の構成成分であるβアクチンまたはγアクチン、リボゾームの構成蛋白質であるＳ２６などの遺伝子が挙げられる。
【００４３】
Ｏｃｔ−３／４遺伝子の発現レベルは、本発明の分化抑制剤に暴露された細胞について決定することができる。分化抑制剤に暴露されていない、即ち、胚性幹細胞から分化誘導されたコントロール細胞のＯｃｔ−３／４遺伝子発現量にくらべ、有意にＯｃｔ−３／４遺伝子の発現量を維持させることができる活性を有する化合物が、胚性幹細胞の未分化を維持する分化抑制剤であるとみなされる。本発明の分化抑制剤を添加して培養した胚性幹細胞のＯｃｔ−３／４遺伝子の発現レベルを評価すると、分化抑制剤を含まない培地にて培養したコントロールの胚性幹細胞にくらべＯｃｔ−３／４遺伝子の発現レベルが有意に高いことが示される。即ち本発明の分化抑制剤が、胚性幹細胞の未分化状態を維持し得ることが確認される。
【００４４】
最適化された胚性幹細胞の未分化維持培養基材をスクリーニングするさらに他の方法として、ステージ特異的胚抗原（Ｓｔａｇｅ Ｓｐｅｃｉｆｉｃ Ｅｍｂｒｙｏｎｉｃ Ａｎｔｉｇｅｎ）（以後ＳＳＥＡと記載）−１、 ＳＳＥＡ−３、 ＳＳＥＡ−４などの未分化な細胞に特異的に発現する抗原を検出方法が挙げられる（Ｓｍｉｔｈら、 Ｎａｔｕｒｅ、 ３３６、 ｐ６８８、 １９８８年、Ｓｏｌｔｅｒら、Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ．Ｕ．Ｓ．Ａ、７５、ｐ５５６５、１９７８年、Ｋａｎｎａｇｉら、ＥＭＢＯ Ｊ．２、ｐ２３５５、１９８３年）。
【００４５】
一つの実施態様において、ＳＳＥＡ−１などの表面抗原は、同抗原を認識する特異的抗体（一次抗体）とインキュベートし、さらに蛍光標識のようなレポーターと結合した第二の抗体（二次抗体）とインキュベートすることにより、標識することができる。この操作により目的の抗原を発現する細胞が、蛍光性になる。次いで、標識された細胞を標準的な方法、例えばフローサイトメーターを用いて、計数、さらには分取され得る。次いで、標識および非標識細胞の数は、目的とする培養基材の効果を決定するために比較され得る。あるいは、非標識細胞表面マーカー抗体に曝露された後、ＥＬＩＳＡ（酵素結合免疫吸着アッセイ）形式において、その細胞は、抗細胞表面抗原抗体（例えば抗ＳＳＥＡ−１抗体）に対して特異的な第二の抗体に曝露され得、そこから、所望の表面抗原を発現する細胞の数が、比色定量的に、または蛍光を測定することにより定量され得る。表面抗原を発現する細胞を定量するさらに他の方法も、細胞培養の当業者に周知である。実施例２に記載の方法により、本発明の分化抑制剤を添加して培養した胚性幹細胞の表面抗原ＳＳＥＡ−１の発現レベルを評価すると、分化抑制剤を含まない培地にて培養したコントロールの胚性幹細胞にくらべ表面抗原ＳＳＥＡ−１の発現レベルが有意に高いことが示される。即ち本発明の分化抑制剤が、胚性幹細胞の未分化状態を維持し得ることが確認される。
【００４６】
本発明によって提供される、幹細胞、または胚性幹細胞の増殖に対する改善された分化抑制剤、培養方法、および培養液は、幹細胞または胚性幹細胞が有用であるすべての技術に対して適用されることが予想される。
【００４７】
本発明の分化抑制剤、培養方法および培養液を用いて産生される細胞は、分化させ、細胞移植に用いたり、人工支持組織の利用と併せ人工的な組織構築に使用され、生体内へ移植したり人工臓器として利用されうる。幹細胞の細胞移植治療や組織工学への利用は、ドナーにおける移植片摘出後の組織欠損やドナー不足など、従来の自家移植を含む移植治療が抱える問題点を解決できる。
【００４８】
本発明の分化抑制剤は、単独または賦形剤あるいは担体と混合して使用することができる。賦形剤および担体としては薬剤学的に許容されるものが選ばれる。例えば液状担体としては水、アルコール類、ジメチルスルフオキシド（ＤＭＳＯ）、もしくは動植物油、合成油などが用いられる。個体担体としては、乳糖、マルトース、シュクロースなどの糖類、アミノ酸類、ヒドロキシセルロースなどのセルロース誘導体、ステアリン酸マグネシウムなどの有機酸塩などが使用される。また、本発明の分化抑制剤の組成中にｐＨ調整等の目的で、酸やアルカリまたは適量の緩衝剤を加えてもよい。
製剤中の本発明における化合物の含量は製剤により種々異なるが、通常０．１〜１００重量％、好ましくは１〜９８％の有効成分を含む。
【００４９】
本発明の分化抑制剤、それを用いた培養方法、それを含む培養液により得られた、非改変および改変された幹細胞、好ましくは胚性幹細胞の培養物は、幹細胞、好ましくは胚性幹細胞のモニタリングまたは幹細胞収集を改良する物質についてスクリーニングするために使用される。例えば、推定の幹細胞或いは胚性幹細胞分化誘導物質は、上記の方法を用いて増殖させた細胞培養物へ添加され得る。推定の幹細胞或いは胚性幹細胞分化誘導物質を欠損した対照細胞培養物と比較して、三胚葉系列への分化を誘導し得る物質は、胚性幹細胞分化誘導因子として同定される。
【００５０】
【実施例】
次に本発明を具体化した実施例を示す。この実施例は、本発明を実施する当業者を補助するために提供される。これらの実施例は、いかなる様式においても、決して本発明の範囲を制限するものではない。
【００５１】
【実施例１】
「ＥＳ細胞培地の作製」
ＥＳ細胞を増殖させる目的で、Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（以下ＤＭＥＭ）（ＧＩＢＣＯ ＢＲＬ社製 Ｃａｔ．Ｎｏ．１１９９５）に、以下に示す最終濃度で因子を添加してＥＳ細胞培地を調製した：１５％ ウシ胎仔血清（ＧＩＢＣＯ ＢＲＬ社製） もしくは１５％ノックアウト血清リプレースメント：ＫＳＲ（ＧＩＢＣＯＢＲＬ社製）、０．１ｍＭ β−メルカプトエタノール（ＳＩＧＭＡ社製）、１×非必須アミノ酸ストック（ＧＩＢＣＯ ＢＲＬ社製 Ｃａｔ．Ｎｏ．１１１４０−０５０）、２ｍＭ Ｌ−Ｇ１ｕｔａｍｉｎｅ（ＧＩＢＣＯ ＢＲＬ社製Ｃａｔ．Ｎｏ．２５０３０−０８１）、１０^３ｕｎｉｔ／ｍｌ ＥＳ ＧＲＯ（ＣＨＥＭＩＣＯＮ Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｉｎｃ．，社製）。ただし、ＥＳ ＧＲＯはマウスＬＩＦを有効成分として含有する。ＥＳ細胞分化抑制アッセイ用の培地として、上記のＥＳ細胞培地からＥＳＧＲＯを除いたアッセイ培地を作製した。
【００５２】
「ＥＳ細胞の培養」
直径５ｃｍの細胞培養用ディッシュに、蒸留水に０．１％の濃度でＧｅｌａｔｉｎ（ＳＩＧＭＡ社製 ＴｙｐｅＡ：ｆｒｏｍ ｐｏｒｃｉｎＥＳｋｉｎ、Ｇ２５００）を溶解し、滅菌した０．１％ゼラチン水溶液５ｍｌを添加し、３７℃で３０分以上静置した。ゼラチン水溶液を除いて、マイトマイシンＣ（協和発酵社製）処理したマウス胚性初代培養細胞（ライフテックオリエンタル社 Ｃａｔ．Ｎｏ． ＹＥ９２８４４００）２×１０^６個を播種し、１０％ウシ胎児血清（ＧＩＢＣＯ ＢＲＬ社製）を含むＤＭＥＭ５ｍｌで、３７℃、５％ＣＯ^２インキュベーター（タバイエスペック社製）で５時間以上培養した。マウス胚性幹細胞株Ｄ３ＥＳ細胞（Ｒｏｌｆ Ｋｅｍｌｅｒ、Ｍａｘ Ｐｌａｎｃｋ Ｉｎｓｔｉｔｕｔｆｕｒ Ｉｍｍｕｎｂｉｏｌｏｇｉｅ、Ｓｔｕｈｅｗｅｇ５１、Ｄ−７９１０８Ｆｒｅｉｂｕｒｇ、Ｇｅｒｍａｎｙ、より入手可能）を、直径５ｃｍのデイッシュに培養した繊維芽細胞フィーダー層上に播種し、５ｍｌのＥＳ培地で、３７℃、５％ ＣＯ_２インキュベーターで２日間培養して増殖させた。
【００５３】
「ＥＳ細胞分化抑制アッセイ１」
前記「ＥＳ細胞の培養」で培養したＤ３ＥＳ細胞をＰＢＳで２回洗浄する。０．２５％トリプシン溶液（ＧＩＢＣＯ ＢＲＬ社製）を加え、３７℃で５分間インキュベートし、未分化のＤ３ＥＳ細胞のコロニーをフィーダーから脱離させた。５ｍｌのＥＳ細胞培地を添加し、小径ピペットを使用して細胞コロニーを分散させ、５０ｍｌの滅菌チューブに移し、卓上遠心機（トミー精工）で１０００ｒｐｍ、約５分間遠心してペレット化した。上清を除き、細胞を２０ｍｌの新鮮なＥＳ細胞培地に再懸濁し、０．１％のゼラチン水溶液で予めコートした直径２０ｃｍの細胞培養用ディッシュに播種し、３７℃で２０分間インキュベートした。同様の操作をもう一度繰り返した。その後、浮遊細胞を含む培地をピペットで回収し、５０ｍｌの滅菌チューブに移し、卓上遠心機で１０００ｒｐｍ、約５分間遠心してペレット化した後、上清を除き、５ｍｌのＥＳ細胞アッセイ培地に再懸濁した。予め０．１％のゼラチン水溶液でコートした９６穴細胞培養用ディッシュ（ＦＡＬＣＯＮ社製 Ｃａｔ．Ｎｏ．３０７２、米国）に、１ウェルあたり３２００個の細胞を、１ウェル当たりのＥＳ細胞アッセイ培地が１００μｌになるように播種した。各ウェルに０．４〜４０μｇ／ｍｌとなるようにジメチルスルフォキシド（ＤＭＳＯ）、または水、或いはその混合物に溶解した本発明の分化抑制剤（Ａ〜Ｅ： ＡはＩＦ ＬＡＢ Ｌｔｄ．社、ウクライナ、Ｂ，ＣはＰＨＡＲＭＥＫＳ社、ロシア、Ｄ，ＥはＳＰＥＣＳ社、オランダから購入）、あるいはＥＳＧＲＯを１０μｌ添加し、３７℃、５％ ＣＯ２インキュベーターで４日間培養した。ＤＭＳＯの培地中への持ち込は最終濃度０．１％以下となるようにした。またコントロールウェルには、ＤＭＳＯのみを最終濃度で０．１％となるように加えた。分化抑制剤Ａ〜Ｅの構造を図１に示した。
【００５４】
「アルカリホスファターゼ定量」
ＥＳ細胞のアルカリホスファターゼ活性を、ｐ−ＮＩＴＲＯＰＨＥＮＹＬ ＰＨＯＳＰＨＡＴＥ ＳＯＬＵＴＩＯＮ（ＭＯＳＳ Ｉｎｃ．社製、ＰＲＯＤＵＣＴ ＮＯ．ＮＰＰＤ−１０００、米国、またはＳＩＧＭＡ社製、Ａ−３４６９、米国製）（以下、ｐ−ＮＰＰ）を用いて定量した。前記「ＥＳ細胞分化抑制アッセイ１」に記載の方法で４日間培養したＥＳ細胞の各ウェルから培地を吸引除去し、その細胞を１００μｌのリン酸緩衝化生理食塩水（ＰＢＳ）で１回洗浄後、ｐ−ＮＰＰ１００μｌを各ウェルに加え、室温で１０分間静置した。各ウェルに１２．５μｌの８Ｍ水酸化ナトリウム溶液を添加し、反応を停止させた。溶液の４０５ｎｍの吸光度（Ｏ．Ｄ．４０５）と６９０ｎｍの吸光度（Ｏ．Ｄ．６９０）を吸光度計（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅｓ社製、型式：ＳＰＥＣＴＲＡ ＭＡＸ１９０）により測定し、Ｏ．Ｄ．４０５−Ｏ．Ｄ．６９０で算出される値をアルカリホスファターゼ活性とした。定量結果をグラフしたものを図２に示した。本発明の分化抑制剤、化合物Ａ〜ＥはコントロールであるＤＭＳＯ（０．１％）に比べて有意にアルカリホスファターゼ活性を増幅させた。即ち、本発明の分化抑制剤は未分化なＥＳ細胞の培養を支持したことがわかる。
【００５５】
【実施例２】
「ＥＳ細胞分化抑制アッセイ２」
前記「ＥＳ細胞の培養」で培養したＤ３ＥＳ細胞をＰＢＳで２回洗浄した。０．２５％トリプシン溶液（ＧＩＢＣＯ ＢＲＬ社製 １５０９０−０４６）を加え、３７℃で５分間インキュベートし、未分化のＤ３ＥＳ細胞のコロニーをフィーダーから脱離させた。５ｍｌのＥＳ細胞培地を添加し、小径ピペットを使用して細胞コロニーを分散させ、５０ｍｌの滅菌チューブに移し、卓上遠心機（トミー精工）で１０００ｒｐｍ、約５分間遠心してペレット化した。上清を除き、細胞を２０ｍｌの新鮮なＥＳ細胞培地に再懸濁し、０．１％のゼラチン水溶液で予めコートした直径１５ｃｍの細胞培養用ディッシュに播種し、３７℃で２０分間インキュベートした。２０分後、浮遊細胞を含む培地をピペットで回収し、再度、０．１％のゼラチン水溶液でコートした直径１５ｃｍの細胞培養用ディッシュに播種し、３７℃で２０分間インキュベートした。２０分後、浮遊細胞を含む培地をピペットで回収し、５０ｍｌの滅菌チューブに移し、卓上遠心機で１０００ｒｐｍ、約５分間遠心してペレット化した後、上清を除き、５ｍｌのＥＳ細胞アッセイ培地に再懸濁した。予め０．１％のゼラチン水溶液でコートした直径１０ｃｍの細胞培養用ディッシュに８×１０^４個の細胞を、ＥＳ細胞アッセイ培地が１０ｍｌになるように播種した。各ディッシュに４０μｇ／ｍｌとなるようにジメチルスルフォキシド（ＤＭＳＯ）、または培地、或いはその混合物に溶解した本発明の分化抑制剤（Ａ〜Ｅ）、あるいはＥＳＧＲＯを１ｍｌ添加し、３７℃、５％ ＣＯ_２インキュベーターで７日間培養した。ＤＭＳＯの培地中への持ち込は最終濃度０．１％以下となるようにした。
【００５６】
「ＳＳＥＡ−１抗原の発現量評価」
前記「ＥＳ細胞分化抑制アッセイ２」で培養した細胞をＰＢＳで２回洗浄し、セルスクレイパーで細胞をディッシュから脱離した。細胞数が６×１０^５個になるように２％ＦＢＳを含むＨａｎｋｓ ｂａｌａｎｃｅｄ ｓａｌｔ ｓｏｌｕｔｉｏｎ（ＨＢＳＳ、Ｉｎｖｉｔｒｏｇｅｎ社製）を３００μｌ加えた。ＨＢＳＳで５０倍希釈したＭＸ−ＳＳＥＡ−１抗体（Ｋｙｏｗａ Ｍｅｄｅｘ社製）を３０μｌ加え４０分間、氷上で静置した。ＨＢＳＳ （１ｍｌ）で二回洗浄後、ＨＢＳＳ （３００μｌ）に再分散し、ＨＢＳＳで２０倍ＦＩＴＣ−Ｇｏａｔ ａｎｔｉ Ｍｏｕｓｅ ＩｇＭ抗体（ＺＹＭＥＤ社製）を３０μｌ加え３０分間、遮光して氷上で静置した。ＨＢＳＳ （１ｍｌ）で二回洗浄後、ＨＢＳＳ （１ｍｌ）に再分散し、２０μｇ／ｍｌ ＰＩ溶液（Ｄｏｊｉｎｄｏ社製）を１００μｌ加え、フローサイトメーター測定に用いるサンプルを得た。またフローサイトメーター測定に用いるサンプルは、凝集塊を除く目的で孔径１００μｍのナイロン・メッシュに通した後に測定を行った。フローサイトメーターはＦＡＣＳＣａｌｉｂｕｒ（ＢＥＣＴＯＮ ＤＩＣＫＩＮＳＯＮ社製、米国）を用い、測定条件データ収集解析ソフトウェアはＣＥＬＬＱｕｅｓｔ（ＢＥＣＴＯＮ ＤＩＣＫＩＮＳＯＮ社製、米国）を用いた。結果を図３に示した。各分化抑制剤を添加して培養した細胞の、１細胞あたりの平均蛍光強度、即ち、１細胞あたりのＳＳＥＡ−１抗原発現強度は、分化抑制剤を含まない培地で培養した細胞にくらべ、有意に亢進していた。また、化合物Ｂ￣Ｅについて同様の実験をしたところ、同様にＳＳＥＡ−１の発現レベルが有意に高いことを示した。即ち、本発明の分化抑制剤は、未分化なＥＳ細胞を維持することが明らかになった。
【００５７】
【実施例３】
「ＳＴＡＴ３活性化アッセイ」
前記「ＥＳ細胞の培養」で培養したＤ３ＥＳ細胞をＰＢＳで２回洗浄する。０．２５％トリプシン溶液（ＧＩＢＣＯ ＢＲＬ社製）を加え、３７℃で５分間インキュベートし、未分化のＤ３ＥＳ細胞のコロニーをフィーダーから脱離させる。５ｍｌのＥＳ細胞培地を添加し、小径ピペットを使用して細胞コロニーを分散させ、 １５ｍｌの滅菌チューブに移し、卓上遠心機（トミー精工）で８００ｒｐｍ、約５分間遠心してペレット化した。上清を除き、細胞を５ｍｌの新鮮なＥＳ細胞培地に再懸濁し、０．１％のゼラチン水溶液で予めコートした直径１０ｃｍの細胞培養用ディッシュに播種し、３７℃で２０分間インキュベートした。２０分後、浮遊細胞を含む培地をピペットで回収し、１５ｍｌの滅菌チューブに移し、卓上遠心機で８００ｒｐｍ、約５分間遠心してペレット化した後、上清を除き、５ｍｌのＥＳ細胞アッセイ培地に再懸濁する。
【００５８】
予め０．１％のゼラチン水溶液でコートした２４穴細胞培養用ディッシュ（ＦＡＬＣＯＮ Ｃａｔ．Ｎｏ．３０４７、米国）に、１ウェルあたり１×１０^５個の細胞を、１ウェル当たりのＥＳ細胞培地が５００μｌになるように播種し、３７℃、５％ ＣＯ_２インキュベーターで１２時間〜２４時間培養した。次ぎに、ＬｉｐｏｆｅｃｔＡＭＩＮＥ ２０００（Ｉｎｖｉｔｒｏｇｅｎ社製、米国）を用いて、添付のプロトコールに従って、ｐＳＴＡＴ３−ＴＡ−Ｌｕｃ、またはｐＴＡ−Ｌｕｃベクター（クロンテック社製、米国）を１ウェルあたり０．９μｇをＥＳ細胞にトランスフェクションした。また、内部コントロールとして、ｐＲＬ−ＴＫベクター（プロメガ社製、米国）を各ウェル０．１μｇ、同時にトランスフェクションした。３７℃、５％ ＣＯ２インキュベーターで４時間培養後、培地を吸引し、ＰＢＳで２回洗浄したのち、ＥＳアッセイ培地を各ウェル５００μｌずつ加えた。次いで、各ウェルに０．４〜４０μｇ／ｍｌとなるようにジメチルスルフォキシド（ＤＭＳＯ）または水或いはその混合物に溶解した分化抑制剤、あるいは１０〜１０，０００ｕｎｉｔ／ｍｌとなるようにＥＳアッセイ培地で希釈したＥＳＧＲＯを５０μｌ添加し３７℃、５％ ＣＯ２インキュベーターで６〜２４時間培養した。続いて、ピッカジーンデュアル・シーパンジー（東洋インキ製造社製、日本）を用いて、添付のプロトコールに従って、各細胞のルシフェラーゼ・レポーター酵素による発光シグナルを測定した。図４に示すように、ＥＳＧＲＯが容量依存的にｐＳＴＡＴ３−ＴＡ−Ｌｕｃによるレポーター酵素の発現を亢進させたのに対し、本発明の化合物Ｂは、レポーター酵素の発現を誘導しなかった。
また、化合物Ａ、Ｃ〜Ｅについて同様の実験をしたが、化合物Ｂを使用したときと同様にレポーター酵素の発現を誘導しなかった。
【００５９】
【図面の簡単な説明】
【図１】本発明の化合物Ａ〜Ｅの構造を示す。
化合物Ａ〜Ｅは、式（Ｉ）のＲ^１〜Ｒ^９が下表に示す基または元素で表される。
【図２】本発明のアルカリホスファターゼ定量の結果を表す。
【図３】本発明のＳＳＥＡ−１抗原の発現量評価における平均蛍光強度の結果を示す。
【図４】本発明のＳＴＡＴ３活性化アッセイの結果を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a stem cell differentiation inhibitor containing a low-molecular compound, particularly an indole derivative or a salt thereof, an stem cell culture method using the same, a culture solution, and a stem cell line prepared using the same.
[0002]
[Prior art]
Organs and tissues damaged by trauma, illness, and aging need to promote regeneration and restore their function. In particular, since organs such as the heart, liver, kidney, and pancreas are essential for life support, their functional decline and abolition are directly linked to death, and transplantation medical treatment for saving lives by organ transplantation is actively performed. However, a new approach is needed to solve this problem due to the permanent shortage of donors.
[0003]
Recently, tissues and organs have been created using stem cells that are present in embryos or adults and are considered to have the ability to divide indefinitely and differentiate in one or more directions. Regenerative medicine is attracting attention as a treatment that surpasses the drawbacks of conventional organ transplantation.
Specifically, stem cells are proliferated and then differentiated and used for cell transplantation, or artificial tissue construction is performed in conjunction with the use of artificial support tissue, which is then transplanted in vivo or used as an artificial organ Things are considered. If stem cells can be used for cell transplantation treatment and tissue engineering, it is expected that problems associated with conventional transplantation treatments including autologous transplantation, such as tissue defects after donor graft removal and donor shortage, are expected.
Stem cells have been identified in various fields such as blood vessels, nerves, blood, cartilage, bone, liver, pancreas, among which totipotent stem cells that have the ability to differentiate into all cell types are the above-mentioned regenerative medicine. In addition to the field, it has attracted particular attention as a cell that can easily provide cells and tissues for use in drug discovery and gene therapy.
As examples of totipotent stem cells, embryonic stem (Embryonic Stem, hereinafter referred to as ES) cells and embryonic germ (Embryonic Germ, hereinafter referred to as EG) cells are known. ES cells are a cell line isolated from the inner cell mass of the mouse blastocyst stage (Inner Cell Mass, ICM) (Evans et al., Nature, 292, p154, 1981). The cells constituting the individual are derived from the inner cell mass (Inner Cell Mass, hereinafter referred to as ICM) in the blastocyst stage or the primary ectoderm derived from the gastrointestinal upper layer (epiblast, hereinafter referred to as epiblast). Can be said to be a totipotent stem cell group. ES cells retain the ability to differentiate into various individual forming tissues, and have the ability to differentiate into any mature adult cell by forming normal and chimeric embryos. It also has the ability to generate various cells such as blood cells, cardiomyocytes, vascular endothelial cells, nerve cells, pigment cells, pancreatic endocrine cells, depending on the differentiation induction conditions in vitro (Toru Nakano, latest medical separate volume-regenerative medicine) , P81-89, 2000).
EG cells are cells established by culturing primordial germ cells in the presence of LIF (Leukemia Inhibitory Factor) and bFGF (basic Fibroblast Growth Factor) (Matsui et al., Cell, 70, p841, n et al., 1992, 1992 et al. Nature, 359, p550, 1992), and has the ability to differentiate into various tissues like ES cells.
Recently, the establishment of ES cell lines other than mice has been reported and shown to have pluripotency similar to mouse ES cells (bovine ES cells: Schellander et al., Theology, 31, p15- 17, 1989, swine ES cells: Strojek et al., Therogenology, 33: p901, 1990, sheep ES cells: Handyside, Roux's Arch. Dev. Biol., 196: p185, 1987, hamster ES cells: Doetschman Biol., 127, p224, 1988, Rhesus monkey ES cells: Thomson et al., Proc. Natl. Acad. Sci. USA, 92, p7844, 1995, Marmoset ES cells: Thomson et al. Biology of Production, 55, p254, 1996 years, human ES cells: Thomson et al., Science, 282, p1145, 1998 years, Reubinoff et al., Nature Biotech, 18, p399, 2000 years).
[0004]
In order to maintain the undifferentiation of ES cells, it is usually necessary to co-culture using fetal fibroblasts as feeder cells. Similar methods have also been used to maintain undifferentiated primate ES cell lines (Thomson et al., Proc. Natl. Acad. Sci. USA, 92, p7844, 1995, Thomson et al., Science, 282, p1145, (1998, Rebinoff et al., Nature Biotech, 18, p399, 2000).
However, the preparation of mouse primary fibroblasts is cumbersome. That is, embryos from 13.5 to 15.5 days are removed from pregnant mice, the embryo body is decomposed by enzyme treatment, and the fibroblasts obtained on the dish are collected. Since it is a primary cell, quality control is complicated, GMP adaptation level management is difficult, and the ability to maintain the undifferentiated state of ES cells may vary depending on the embryo body used. As an ES cell culture method that does not go through this complicated preparation work, there is a method using STO cells (ATCC 56-X) which is a cell line of mouse embryo fibroblasts. However, the ability of STO cells to maintain undifferentiated ES cells is likely to change, and mouse primary fibroblasts are superior for stable culture of ES cells.
Recently, cases of infection with endogenous viruses between different animals have been reported (van der Laan et al., Nature, 407, p90, 2000). In the culture method aiming at the utilization of human ES cells for medical use, development of a culture method that avoids contact between different animal cells as much as possible is desired. Therefore, the above ES cell undifferentiated maintenance culture method using mouse-derived cells is not suitable for the culture of ES cells for medical purposes.
As a method for culturing primate ES cells that do not use mouse-derived feeder cells, a method of adding secreted components of mouse primary fibroblasts to the culture medium has been reported (see, for example, Patent Document 1). However, even in this case, ES cells in culture are exposed to unidentified factors secreted from mouse cells. Therefore, ES cells cultured in such an environment are not suitable for medical use. In addition, there is a risk of infection with endogenous viruses. Accordingly, the disadvantages caused by co-culture with mouse primary fibroblasts have not been solved at all.
As an undifferentiated maintenance culture method for mouse-derived feeder cells and mouse ES cells that do not use mouse-feeder cell-derived secretory components, a culture method using a gelatin-coated culture dish is already known. It is essential to add a factor (leukemia inhibitory factor, LIF) to the medium (for example, see Non-Patent Document 1). . Since LIF is a cytokine, it has problems such as high cost and storage stability and is not suitable for mass culture. In addition, the effect of LIF is limited to ES cells derived from very specific mouse strains (129 / sv strain and C57BL / 6 strain), and no significant effect is seen in other species. In particular, in primate ES cells, it has been clarified that an undifferentiated state cannot be maintained only by adding LIF to the medium (see, for example, Non-Patent Documents 2 and 3).
[0005]
Therefore, there has never been a differentiation inhibitor that makes it possible to cultivate totipotent stem cells safely and in large quantities at low cost, and methods for culturing totipotent stem cells at low cost safely and in large quantities are also known. There wasn't.
Although the differentiation inhibitor of the present invention contains a low molecular compound as an active ingredient, it has not been known so far that the low molecular compound maintains the undifferentiated state of totipotent stem cells. Therefore, the action of maintaining the undifferentiation of totipotent stem cells possessed by the low molecular weight compound represented by the general formula (I) has not been known at all.
[0006]
[Patent Document 1]
JP 2001-17163 A
[Non-Patent Document 1]
Smith et al., Dev. Biol. 121, p1, 1987
[Non-Patent Document 2]
Thomson et al., Proc. Natl. Acad. Sci. USA, 92, p7844, 1995,
[Non-Patent Document 3]
Thomson et al., Science, 282, p1145, 1998
[0007]
[Problems to be solved by the invention]
An object of the present invention is to provide a differentiation inhibitor capable of culturing stem cells or embryonic stem cells in an undifferentiated state without using feeder cells or feeder cell-derived components. Another object of the present invention is to provide a method for culturing stem cells or embryonic stem cells in an undifferentiated state using such a differentiation inhibitor, without using feeder cells or feeder cell-derived components. It is to provide a cell culture medium containing a different differentiation inhibitor and to provide a cell line produced by culturing using such a differentiation inhibitor.
[0008]
[Means for Solving the Problems]
The present invention has been made to achieve the above-described problem, and includes a differentiation inhibitor capable of culturing stem cells and embryonic stem cells in an undifferentiated state, a stem cell using the same, and a method for culturing embryonic stem cells, The present invention relates to a cell culture medium containing such a differentiation inhibitor, and a cell line produced by culturing using such a differentiation inhibitor.
[0009]
That is, the present invention relates to a stem cell differentiation inhibitor containing a low molecular compound, particularly an indole derivative, as an active ingredient.
Examples of the indole derivative in the present invention include compounds represented by the following formula (I).
[0010]
[Chemical 3]

[0011]
(Wherein R ¹ ~ R ⁹ Represents the same or different electron-withdrawing group, electron-donating group or hydrogen electron. Specifically, an alkyl group, an alkoxy group, a hydroxyl group, a nitro group, an arylaminovinyl group, a hydrogen atom and the like are exemplified.
[0012]
Furthermore, the present invention relates to a method for culturing stem cells, particularly embryonic stem cells, in an undifferentiated state using the aforementioned stem cell differentiation inhibitor.
The present invention also relates to a culture solution for stem cells using the aforementioned stem cell differentiation inhibitor.
Furthermore, this invention relates to the cell produced by culture | cultivating using the said stem cell differentiation inhibitor.
[0013]
According to the present invention, stem cells, preferably embryonic stem cells, can be proliferated in large quantities and safely in an undifferentiated manner, for an extended period or indefinitely. The differentiation-suppressing agent provided by the present invention, a culture method using the same, and a culture solution can be applied to produce stem cells, preferably embryonic stem cells, as a cell source used for cell transplantation. In addition to the many benefits obtained by utilizing stem cells, preferably embryonic stem cells, the differentiation-suppressing agents provided by the present invention and the culture methods using them are embryos having one or many genetic modifications. It can be applied to produce sex stem cells. Examples of such applications include, but are not limited to, the development of cell-based models for disease as well as the development of tissues that are specific for transplantation to treat genetic diseases.
[0014]
The following terms are defined as provided in this section unless stated otherwise. All other terms used herein are defined with respect to their terminology in the particular field relating to that term, unless stated otherwise.
Stem cell: A stem cell refers to another cell type with a specific function, ie, a cell that can differentiate into a terminally differentiated cell or other stem cell type that can differentiate into a narrower range of cell types. .
Totipotent stem cells: Totipotent stem cells are cells that can differentiate into any cell type, including pluripotent cells and fully differentiated cells (ie, cells that can no longer differentiate into various cells). Say that.
Pluripotent stem cell: A pluripotent stem cell refers to a cell that is not necessarily of all types, but can differentiate into one of a number of different cell types. One example of a pluripotent cell is a bone marrow stem cell, which can differentiate into various blood cell types, such as lymphocytes and red blood cells, other than neurons. Thus, it is recognized that all totipotent cells are pluripotent, whereas not all pluripotent cells are totipotent.
Embryonic stem cells: Embryonic stem cells are totipotent cells obtained from the morula or blastocyst stage of a stem cell, especially an embryo at the pre-implantation stage, and are also called ES cells. In addition, embryonic stem cells may refer to pluripotent stem cells derived from primordial germ cells of embryos or fetuses (fetuses) that are determined to be sperm or eggs. However, this cell is sometimes called an embryonic germ (EG) cell and is sometimes distinguished from an embryonic stem cell. The embryonic stem cells used in the present specification may be of any animal species, and examples include embryonic stem cells of primates including humans, mammals other than primates, birds and the like.
Totipotency: Totipotency refers to a state that can differentiate into any cell type, including pluripotent cells and fully differentiated cells (ie, cells that can no longer differentiate into various cells).
Pluripotency: Pluripotency refers to a condition that can differentiate into one of a number of different cell types, although not necessarily all types.
Undifferentiated: Undifferentiated is a cell in which one cell or an arbitrary cell population composed of a plurality of cells is capable of differentiating into one or more further differentiated cells, Or it is the state which is a cell population containing this cell.
Feeder: A feeder used for the purpose of describing the present invention refers to one in which totipotent stem cells are plated thereon and provides an environment that aids in the growth of the plated totipotent stem cells.
Feeder cells: Feeder cells used for purposes of describing the present invention are non-totipotent stem cells on which totipotent stem cells are plated, and non-totipotent stem cells are a proliferation of plated totipotent stem cells. Providing an environment that helps.
Cell-derived components: All components derived from cells such as components secreted from cells, contents, and cell membrane components.
[0015]
The present invention relates to a differentiation inhibitor capable of proliferating and maintaining stem cells, preferably embryonic stem cells in an undifferentiated state, a culture method using the same, a culture solution using the same, and a culture medium using the same. A cell line is provided. The differentiation-suppressing agent, culture method and culture solution provided in the present invention proliferate and maintain undifferentiated embryonic stem cells more simply and safely than before. The cell culture method comprising the differentiation inhibitor of the present invention can also be used to screen for specific differentiation inducers and useful combinations of differentiation inducers. The ability to grow undifferentiated embryonic stem cells using the differentiation inhibitors and culture methods of the present invention produces embryonic stem cell lines with single or multiple genetic modifications that have important therapeutic applications. Provide important benefits including capabilities.
[0016]
The differentiation inhibitor of the present invention can be used as long as it is a chemically stable low molecular weight compound and has an activity of maintaining stem cells in an undifferentiated state, and an indole derivative is preferable. More preferably, an indole derivative having a structure represented by the formula (I), a salt thereof, or an ester compound is desirable. Where R ¹ ~ R ⁹ As, examples thereof include the same or different electron-withdrawing groups, groups selected from electron-donating groups or hydrogen atoms, or atoms. The electron donating group is a substituent capable of donating electrons to the benzene ring, and the electron withdrawing group is a substituent having a property of attracting π electrons on the benzene ring. It is also possible to define σ <0 as an electron-donating group and σ> 0 as an electron-withdrawing group using Hammett's substituent constant σ (Basic Organic Reaction Theory, written by Shizunobu Hashimoto, Sankyo Publishing, 1997). . More preferably, R ¹ ~ R ⁹ Are the same or different, alkyl group, alkenyl group, alkynyl group, phenyl group, naphthyl group, furyl group, thienyl group, alkoxy group, alkylamino group, alkylcarbonyl group, benzoyl group, naphthoyl group, furoyl group, thenoyl group A group selected from the group consisting of a dialkylcarbamoyl group, an acetyl group, a butanoyl group, a methoxycarbonyl group, a cycloalkyl group, a benzyloxy group, an adamantyloxy group, an arylaminovinyl group, a hydroxyl group and a nitro group, or a halogen atom or hydrogen An atom etc. are mentioned. Furthermore, R ¹ ~ R ⁹ Is preferably a group or an atom selected from the group consisting of an alkyl group, an alkoxy group, a hydroxyl group, a nitro group, an arylaminovinyl group, and a hydrogen atom.
[0017]
Of these compounds, the present invention particularly preferably uses an indole derivative represented by the following formula (II) or a salt thereof.
[Formula 4]

A ¹ ~ A ³ Represents a lower alkyl group, a lower alkoxy group, or a hydrogen atom, which may be the same, and A ⁴ Represents a lower alkyl group or an arylaminovinyl group.
[0018]
In compound (II), A ¹ ~ A ⁴ Examples of the lower alkyl group represented by are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like. Particularly preferred is methyl.
[0019]
In compound (II), A ¹ ~ A ⁴ Examples of the lower alkoxy group represented by: include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like, and methoxy is particularly preferable.
[0020]
In compound (II), A ⁴ As the arylaminovinyl group represented by the formula (III), it is desirable to use a group represented by the formula (III). Examples of Ar (aryl group) represented by the formula (III) include phenyl group, tolyl group, methoxyphenyl group, xylyl group, naphthyl group, anthryl group, etc., and 4-methoxyphenyl group is particularly preferable. It is.
[Chemical formula 5]

[0021]
The salt of compound (II) is preferably a pharmaceutically acceptable salt. For example, hydrochloride, sulfate, phosphate, hydrobromide, acetate, maleate, fumarate, succinate, methanesulfonate, p-toluenesulfonate, citrate, tartaric acid A salt or the like may be formed.
[0022]
Examples of the ester compound of Compound I or II include carboxylic acid ester, sulfonic acid ester, and inorganic acid ester.
[0023]
The concentration of the differentiation inhibitor of the present invention is desirably used in the range of 0.1 ng / ml to 1 mg / ml, preferably 10 ng / ml to 100 μg / ml, more preferably 100 ng / ml to 10 μg / ml. It is to be used in a range.
[0024]
The differentiation inhibitor of the present invention includes a low molecular weight compound that maintains undifferentiation of embryonic stem cells without activation of STAT3 (signal transducer and activator of transcription 3), particularly an isoquinoline derivative. Embryonic stem cells of a specific mouse lineage are maintained undifferentiated by LIF regardless of the presence or absence of feeder cells composed of fibroblasts derived from mouse embryos. LIF is known to signal downstream through activation of STAT3 (Matsuda et al., EMBO Journal, 18, 15, p4261, 1999). However, since it has recently been reported that there is a differentiation inhibitor that maintains undifferentiation of embryonic stem cells without activating STAT3 (Dani et al., Developmental Biology, 203, p149, 1998), It is believed that there is a mechanism for maintaining undifferentiated embryonic stem cells that does not involve STAT3. Furthermore, LIF has no effect on maintaining undifferentiated primate embryonic stem cells (Thomson et al., Proc. Natl. Acad. Sci. USA, 92, p7844, 1995, Thomson et al., Science, 282, p1145. , 1998, Rebinoff et al., Nature Biotech, 18, p399, 2000), suggesting that primate embryonic stem cells have a mechanism that maintains an undifferentiated state with signals different from LIF and STAT3. The differentiation inhibitor of the present invention includes a low molecular weight compound that maintains undifferentiation of embryonic stem cells without activating STAT3. In other words, a low molecular weight compound having an activity of maintaining undifferentiation of embryonic stem cells by an action different from that of LIF is included.
[0025]
Further, according to a recent report, in some types of bone marrow-derived mesenchymal stem cells, as in the case of embryonic stem cells, the culture of mouse-derived stem cells is LIF-dependent, but human-derived stem cells are independent of LIF. It is shown that. (Verfaillie et al., Nature, 418, p41, 2002) This suggests that pluripotent stem cells have the same mechanism of maintaining undifferentiation as embryonic stem cells, and thus in primate stem cells As with embryonic stem cells, it is suggested that undifferentiation may be maintained by a signal different from the LIF-STAT3 pathway. The differentiation inhibitor of the present invention includes a low molecular weight compound having an activity of maintaining an undifferentiated state of a cell without using STAT3.
[0026]
The differentiation inhibitor of the present invention can be used by adding to any mammalian cell culture basal medium that is a basal medium for animal cell culture. Examples of animal cell basal medium include, but are not limited to, Dulbecco's modified Eagle medium: DMEM, knockout DMEM, Glasgow MEM: GMEM, RPMI 1640, IMDM (GIBCO, USA). In one embodiment, the cell culture medium is Dulbecco's Modified Eagle Medium (DMEM). Furthermore, serum or various growth factors as serum substitutes can be added to these basal media for use. The serum can be any serum or serum-based solution that provides nutrients that are effective in maintaining the growth and viability of stem cells and embryonic stem cells. Examples of such sera include fetal calf serum (FCS), bovine serum (CS), horse serum (HS), etc., and serum substitutes well known to those skilled in the art, proteins, amino acids, lipids Vitamins can be used alone or in combination. Proteins include insulin, transferrin, albumin, peptone, FGF (Fibroblast Growth Factor), EGF (Episial Growth Factor), etc., and amino acids include arginine, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, alanine, methionine, Examples of vitamins include, but are not limited to, pantothenic acid, choline, folic acid, inositol, nicotinic acid amide, riboflavin, thiamine, and pyridoxine. In one embodiment, the serum is fetal bovine serum. In a more particular embodiment, fetal calf serum is provided at a concentration between about 25% and about 1%. In an even more specific embodiment, the fetal calf serum concentration in the cell culture medium is 15%. In another embodiment, the serum replacement is knockout serum replacement: KSR (GIBCOBRL, USA).
[0027]
The cell culture medium also contains an antioxidant (reducing agent) (eg, β- <mercaptoethanol). In certain preferred embodiments, β- <mercaptoethanol has a concentration of about 0.1 mM. Other antioxidants (eg monothioglycerol or dithiothreitol (DTT) alone or in combination) can be used for similar effects. Still other equivalent materials are well known to those skilled in the art of cell culture.
[0028]
The differentiation inhibitor of the present invention and its active ingredient can be used together with any culture substrate or fixed to the culture substrate. A porous material can be used as the culture substrate. The porous body refers to a substrate having a large number of fine pores, and the material, thickness, shape, dimensions and the like are not particularly limited. The material of the porous body may be an organic material, an inorganic material, or a composite material composed of an organic material and an inorganic material. The shape of the porous body may be any of a flat plate shape, a spherical shape, a rod shape, a fiber shape, and a hollow shape. Examples thereof include yarn, hollow fiber, and particle. In culturing cells, a non-woven fabric is more preferable in consideration of the ability to easily control the size of the holes that support the cells so that they can be cultivated three-dimensionally, the ease of manufacturing the substrate, the cost, and the like. The pore size of the porous body is not particularly limited, but considering that the cells can be supported three-dimensionally, the average pore diameter is preferably 0.1 μm or more and 100 μm or less, and preferably 1 μm. More preferably, it is 50 μm or less. The fiber diameter is not particularly limited, but is preferably 0.03 denier or less.
[0029]
The porous body may be subjected to a surface coating treatment with a polymer compound in order to improve cell adhesion, differentiation maintenance function, and proliferation ability. The polymer substance means a substance having a molecular weight of several hundreds or more in which monomers of one or more kinds of repeating structural units are linked one-dimensionally, two-dimensionally and three-dimensionally. Polymer materials can be broadly classified into three categories: natural polymer materials, semi-synthetic polymer materials, and synthetic polymer materials, and any polymer material can be used in the present invention.
[0030]
For example, natural polymer substances include mica (mica), asbestos (asbestos), graphite (graphite), saccharides such as diamond, starch, cellulose, and alginic acid, and proteins such as gelatin, fibronectin, and fibrinogen. Is mentioned. Examples of the semi-synthetic polymer material include glass, cellulose nitrate, cellulose acetate, hydrochloric acid rubber, carboxymethyl cellulose and the like. Synthetic polymer materials include polyphosphonitrile chloride, polyethylene, polyvinyl chloride, polyamide, polyethylene terephthalate, polysulfone, polyacrylonitrile, polyvinyl alcohol, polymethyl methacrylate, polyhydroxyethyl methacrylate, polydimethylaminoethyl methacrylate and hydroxyethyl methacrylate. Examples thereof include a copolymer composed of two or more kinds of synthetic monomers represented by a copolymer of dimethylaminoethyl methacrylate.
Considering the ease of coating treatment, organic polymer materials are preferable, and proteins and peptides, and organic synthetic polymer materials are more preferable.
[0031]
By using a culture substrate on which the differentiation inhibitor of the present invention is immobilized as a cell-capturing material, a method and an apparatus capable of separating and culturing stem cells or embryonic stem cells from a population consisting of a plurality of different cells are provided. obtain. That is, a cell-containing solution containing stem cells or embryonic stem cells and cells to be removed is introduced into a container filled with a cell capturing material made of a culture substrate such as a porous body, to which the differentiation inhibitor of the present invention is fixed. The stem cell or embryonic stem cell culturing method is characterized in that stem cells or embryonic stem cells are captured by a cell trapping material, the cells to be removed are led out of the container, and then cultured together with the container, and the differentiation inhibitor of the present invention A cell culture apparatus in which a container is filled with a cell capturing material comprising a culture substrate on which is immobilized, wherein the cell capturing material can be used as a cell culture carrier, and the container can be used for cell culture. It is a cell culture device characterized by being. The cells to be removed refer to all cells other than stem cells or embryonic stem cells. This also includes cells that have differentiated from stem cells or embryonic stem cells and have lost pluripotency. The cell-containing solution to be introduced into the cell trapping material may be any cell solution containing stem cells or embryonic stem cells. For example, blood, bone marrow, debris tissue fluid, or stem cell or embryonic stem cell culture solution Etc.
[0032]
The present invention relates to a method for expanding stem cells as well as embryonic stem cells. A culture of stem cells and embryonic stem cells grown using the differentiation inhibitor of the present invention can be provided.
[0033]
The cells cultured using the differentiation inhibitor of the present invention include all stem cells and embryonic stem cells that can be obtained using known methods and materials. Examples of stem cells include stem cells that can be obtained by the following known methods. Bone marrow cells ("Bone marrow transplantation guide" HJ Dieg, HG Klingemann, GL Phillips, translated by Shinhei Kasakura), bone marrow stem cells (Osawa et al., Science, 273, p242-245, 1996, Goodell) JE Med. 183, p1797-1806, 1996, Verfaillie et al., Nature, 418, p41, 2002), neural stem cells (Reynolds et al., Science, p1707-1710, 1992), tissue stem cells (Goodell) JE Med. 183, p1797-1806, 1996, Matsuzaki et al., Experimental Medicine, 19, p345-349, 2001, Blau et al., Cell, 105, p829-841, 2001), mesenchymal system Stem cells (Liechty et al., Nature Medicine, 6, p1282-1286, 2000 years, Pittenger et al., Science, 284, p143-147, 1999 years).
[0034]
Further, embryonic stem cells to be cultured can be obtained using known methods and materials shown below. Mouse embryonic stem cells: Evans et al., Nature, 292, p154, 1981, bovine ES cells: Schellander et al., Therology, 31, p15-17, 1989, porcine ES cells: Strokek et al., Therogenology, 33: p901, 1990 Sheep ES cells: Handyside, Roux's Arch. Dev. Biol. 196: p185, 1987, hamster ES cells: Doetschman et al., Dev. Biol. , 127, p224, 1988, Monkey ES cells: Thomson et al., Proc. Natl. Acad. Sci. USA, 92, p7844, 1995, human ES cells: Thomson et al., Science, 282, p1145, 1998, Rebinoff et al., Nature Biotech, 18, p399, 2000, human EG cells: Garhart et al., Proc. Natl. Acad. Sci. USA, 95, p13726, 1998. Mouse embryonic stem cells (129SV and C57 / BL6) can be purchased from Dainippon Pharmaceutical.
[0035]
The differentiation inhibitor provided by the present invention can be used for all stem cells and embryonic stem cells, but is preferably used for mammalian stem cells and embryonic stem cells, and is preferably used for primate stem cells and embryonic stem cells. preferable.
[0036]
Once isolated, stem cells as well as embryonic stem cells can be cultured in an undifferentiated state using the differentiation inhibitor of the present invention.
[0037]
The degree of undifferentiation of stem cells, preferably embryonic stem cells, cultured using the differentiation inhibitor of the present invention can be confirmed by measuring alkaline phosphatase (ALP) activity present in the cell membrane of the stem cells. It is known that ALP activity is maintained in undifferentiated embryonic stem cells and decreases upon differentiation (Williams et al., Nature, 336, pp684, 1988, Thomson et al., Science, 282, p1145, 1998). Examples include a method of detecting alkaline phosphatase (ALP) activity by a staining method using an insoluble substrate or a spectroscopic measurement method using a water-soluble substrate.
[0038]
In one embodiment, ALP activity can be quantified by spectroscopic methods. An alkaline solution of paranitrophenyl phosphate (pNPP) is added to the cells on the culture dish. PNPP is hydrolyzed by ALP present on the cell membrane to produce paranitrophenol. By measuring the absorbance of the resulting solution at 405 nm, alkaline phosphatase activity can be quantified. When the ALP activity of embryonic stem cells cultured by adding the differentiation inhibitor of the present invention was measured by the method described in Example 4, it was significantly higher than that of control embryonic stem cells cultured in a medium not containing the differentiation inhibitor. Are shown to have high ALP activity. That is, it is confirmed that the differentiation inhibitor of the present invention can be proliferated while maintaining the undifferentiated state of embryonic stem cells.
[0039]
In other embodiments, ALP activity can also be detected by ALP staining. A reaction solution containing a phosphate ester salt and a diazonium salt as a substrate is added to the cells on the culture dish. Phosphate ester salt is hydrolyzed by alkaline phosphatase present in the form of a cell membrane, and then coupled with a diazonium salt to produce an azo dye, which precipitates at the ALP active site. By counting the number of stained colonies, the ALP activity of the cells can be quantified, that is, the degree of undifferentiation of the cells can be quantified. When embryonic stem cells cultured with the differentiation inhibitor of the present invention are stained with ALP, it is shown that the ALP activity is significantly higher than that of control cells without the differentiation inhibitor, that is, the differentiation inhibitor of the present invention is an embryo. It has been confirmed that it can proliferate while maintaining the undifferentiated state of sex stem cells.
The
[0040]
In addition, the degree of embryonic stem cell undifferentiation can be confirmed by measuring the expression level of the Oct-3 / 4 gene. The Oct-3 / 4 gene is a transcription factor belonging to the POU family and is specifically expressed in embryonic stem cells and embryonic cancer cells (EC cells) in an undifferentiated state (Okamoto et al., Cell, 60, p461, 1990). In embryogenesis, it is expressed only in the undifferentiated cell lineage (Scholer, Trends Genet, 7, p323, 1991). Furthermore, homozygotes of Oct-3 / 4 gene-disrupted mice stop development at the blastocyst stage. Thus, it has been revealed that it has an important function for maintaining an undifferentiated state (Nichols et al., Cell, 95, p379, 1998). On the other hand, it has recently been clarified that overexpression of the Oct-3 / 4 gene promotes differentiation of embryonic stem cells (Niwa et al., Nat. Genet. 24, p372, 2000). Maintaining the expression level within a certain range is important for maintaining an undifferentiated state. As one embodiment for measuring the expression level of the Oct-3 / 4 gene, a quantitative PCR (polymerase chain reaction) method can be used.
[0041]
In one embodiment, a real-time PCR method is used, and a simple and reliable quantitative measurement is possible with a wide dynamic range. Real-time PCR techniques include a method using a Taq Man probe using ABI PRISM7700 ™ (Applied Biosystems) and a method using LightCycler ™ (Roche Diagnostics). In particular, in the latter case, a change in amplification amount of DNA synthesized in each cycle can be detected in real time under a high-speed reaction cycle in which the PCR temperature cycle is completed in several tens of minutes. As a DNA detection method of the real-time PCR method, there are four methods using a DNA binding dye (intercalator), a hybridization probe (kissing probe), a TaqMan probe, and a Sunrise uniprimer (molecular beacon). In addition, the expression level of the Oct-3 / 4 gene can be analyzed using a DNA binding dye such as SYBR GreenI. SYBR Green I is a double-stranded DNA-specific binding dye, and the original fluorescence intensity is enhanced by binding to the double strand. If SYBR Green I is added during the PCR reaction and the fluorescence intensity is measured at the end of each cycle of the extension reaction, an increase in the PCR product can be detected. In order to detect the Oct-3 / 4 gene, primers are designed using commercially available gene analysis software or the like based on the sequence of the Oct-3 / 4 gene in the same manner as in ordinary PCR. Since SYBR Green I also detects non-specific products, it is necessary to set optimal primers. As design criteria, attention should be paid to the length of the oligomer, the base composition of the sequence, the GC content, and the Tm value.
[0042]
In many cases, it is the amount of DNA of interest per fixed amount of sample that is intended to be revealed in quantitative PCR. For this purpose, it is necessary to first evaluate the amount of sample added to the reaction system. In this case, another DNA serving as an internal standard reflecting the sample amount can be measured separately from the target DNA, and the sample amount initially added to the reaction system can be corrected. As an internal standard used for the purpose of correcting the sample amount, a housekeeping gene that is considered to have no difference in expression level depending on tissues can be used. Examples include genes such as glyceroaldehyde phosphate dehydrogenase (GAPDH), which is a major glycolytic enzyme, β-actin or γ-actin, which is a constituent component of the cytoskeleton, and S26, which is a constituent protein of ribosome.
[0043]
The expression level of the Oct-3 / 4 gene can be determined for cells exposed to the differentiation inhibitor of the present invention. It is possible to maintain the Oct-3 / 4 gene expression level significantly compared to the Oct-3 / 4 gene expression level of control cells that have not been exposed to a differentiation inhibitor, that is, differentiation-induced from embryonic stem cells. Compounds with activity are considered to be differentiation inhibitors that maintain undifferentiated embryonic stem cells. When the expression level of the Oct-3 / 4 gene in the embryonic stem cells cultured with the differentiation inhibitor of the present invention added was evaluated, Oct-3 compared to the control embryonic stem cells cultured in a medium containing no differentiation inhibitor. It is shown that the expression level of / 4 gene is significantly high. That is, it is confirmed that the differentiation inhibitor of the present invention can maintain the undifferentiated state of embryonic stem cells.
[0044]
As yet another method for screening an undifferentiated maintenance culture substrate for optimized embryonic stem cells, a stage-specific embryonic antigen (hereinafter referred to as SSEA) -1, SSEA-3, SSEA-4 (Smith et al., Nature, 336, p688, 1988, Solter et al., Proc. Natl. Acad. Sci. USA, 75). , P 5565, 1978, Kannagi et al., EMBO J. 2, p 2355, 1983).
[0045]
In one embodiment, a surface antigen such as SSEA-1 is incubated with a specific antibody that recognizes the same antigen (primary antibody), and further a second antibody (secondary antibody) conjugated to a reporter such as a fluorescent label. Incubation with can be labeled. By this operation, cells expressing the target antigen become fluorescent. The labeled cells can then be counted and even sorted using standard methods, such as a flow cytometer. The number of labeled and unlabeled cells can then be compared to determine the effect of the intended culture substrate. Alternatively, after exposure to an unlabeled cell surface marker antibody, in an ELISA (enzyme linked immunosorbent assay) format, the cell is a second antibody specific for an anti-cell surface antigen antibody (eg, an anti-SSEA-1 antibody). From which the number of cells expressing the desired surface antigen can be quantified colorimetrically or by measuring fluorescence. Still other methods for quantifying cells expressing surface antigens are well known to those skilled in the art of cell culture. When the expression level of the surface antigen SSEA-1 of embryonic stem cells cultured by adding the differentiation inhibitor of the present invention was evaluated by the method described in Example 2, the control cultured in a medium containing no differentiation inhibitor was used. It is shown that the expression level of the surface antigen SSEA-1 is significantly higher than embryonic stem cells. That is, it is confirmed that the differentiation inhibitor of the present invention can maintain the undifferentiated state of embryonic stem cells.
[0046]
The improved differentiation-suppressing agent, culture method, and culture medium for the proliferation of stem cells or embryonic stem cells provided by the present invention are applicable to all techniques in which stem cells or embryonic stem cells are useful. Is expected.
[0047]
Cells produced using the differentiation inhibitor, culture method and culture solution of the present invention are differentiated and used for cell transplantation or used for artificial tissue construction together with the use of artificial support tissue, and transplanted in vivo. And can be used as an artificial organ. The use of stem cells for cell transplantation treatment and tissue engineering can solve the problems of conventional transplantation treatments including autologous transplantation, such as tissue loss after donor graft removal and donor shortage.
[0048]
The differentiation inhibitor of the present invention can be used alone or mixed with an excipient or a carrier. As the excipient and carrier, pharmaceutically acceptable ones are selected. For example, water, alcohols, dimethylsulfoxide (DMSO), animal or vegetable oils, synthetic oils, etc. are used as the liquid carrier. As the solid carrier, saccharides such as lactose, maltose and sucrose, amino acids, cellulose derivatives such as hydroxycellulose, and organic acid salts such as magnesium stearate are used. Moreover, you may add an acid, an alkali, or a suitable amount of buffering agents in the composition of the differentiation inhibitor of this invention for the purpose of pH adjustment.
The content of the compound of the present invention in the preparation varies depending on the preparation, but usually contains 0.1 to 100% by weight, preferably 1 to 98% of the active ingredient.
[0049]
The differentiation-suppressing agent of the present invention, a culture method using the same, and a culture of non-modified and modified stem cells, preferably embryonic stem cells, obtained by a culture medium containing the same, are stem cells, preferably embryonic stem cells. Used to screen for substances that improve monitoring or stem cell collection. For example, a putative stem cell or embryonic stem cell differentiation inducer can be added to a cell culture grown using the method described above. A substance capable of inducing differentiation into three germ layers is identified as an embryonic stem cell differentiation inducing factor as compared to a control cell culture lacking a putative stem cell or embryonic stem cell differentiation inducing substance.
[0050]
【Example】
Next, examples embodying the present invention will be shown. This example is provided to assist one of ordinary skill in practicing the present invention. These examples in no way limit the scope of the invention in any way.
[0051]
[Example 1]
"Preparation of ES cell medium"
For the purpose of growing ES cells, an ES cell medium was prepared by adding factors to Dulbecco's Modified Eagle Medium (hereinafter DMEM) (Cib. No. 11995, manufactured by GIBCO BRL) at the following final concentrations: 15 % Fetal bovine serum (GIBCO BRL) or 15% knockout serum replacement: KSR (GIBCOBRL), 0.1 mM β-mercaptoethanol (SIGMA), 1 × non-essential amino acid stock (GIBCO BRL Cat. No. 11140-050), 2 mM L-G1 utamine (Cib. No. 25030-081 manufactured by GIBCO BRL), 10 ³ unit / ml ES GRO (manufactured by Chemicon International Inc., Inc.). However, ES GRO contains mouse LIF as an active ingredient. As a medium for ES cell differentiation inhibition assay, an assay medium in which ESGRO was removed from the above ES cell medium was prepared.
[0052]
“Culture of ES cells”
Gelatin (Type A: from porcin ESkin, G2500, manufactured by SIGMA) was dissolved in distilled water at a concentration of 0.1% in a cell culture dish having a diameter of 5 cm, and 5 ml of a sterilized 0.1% gelatin aqueous solution was added thereto at 37 ° C. And left for at least 30 minutes. Mouse embryonic primary cultured cells treated with mitomycin C (manufactured by Kyowa Hakko) except for an aqueous gelatin solution (Lifetech Oriental Cat. No. YE9284400) 2 × 10 ⁶ 5% DMEM containing 10% fetal calf serum (GIBCO BRL), 37 ° C, 5% CO ² The cells were cultured for 5 hours or more in an incubator (manufactured by Tabai Espec). Mouse embryonic stem cell line D3ES cells (Rolf Kemler, Max Plank Institute Institute Immunobiology, Stuweweg 51, available from D-79108 Freiburg, Germany) are seeded on a 5 cm diameter fibroblast feeder layer on a 5 cm ES fibroblast feeder layer. In medium, 37 ° C, 5% CO ₂ The cells were grown for 2 days in an incubator.
[0053]
"ES cell differentiation inhibition assay 1"
The D3ES cells cultured in the “ES cell culture” are washed twice with PBS. A 0.25% trypsin solution (GIBCO BRL) was added and incubated at 37 ° C. for 5 minutes, and undifferentiated D3ES cell colonies were detached from the feeder. 5 ml of ES cell medium was added, cell colonies were dispersed using a small-diameter pipette, transferred to a 50 ml sterile tube, and pelleted by centrifugation at 1000 rpm for about 5 minutes in a tabletop centrifuge (Tomy Seiko). The supernatant was removed and the cells were resuspended in 20 ml of fresh ES cell medium, seeded in a 20 cm diameter cell culture dish pre-coated with 0.1% aqueous gelatin and incubated at 37 ° C. for 20 minutes. The same operation was repeated once more. Thereafter, the medium containing the floating cells is collected with a pipette, transferred to a 50 ml sterilized tube, pelleted by centrifugation at 1000 rpm for about 5 minutes in a desktop centrifuge, the supernatant is removed, and the suspension is resuspended in 5 ml of ES cell assay medium. It became cloudy. In a 96-well cell culture dish (FALCON Cat. No. 3072, USA) previously coated with a 0.1% gelatin aqueous solution, 3200 cells per well and 100 μl of ES cell assay medium per well Sowing. Differentiation inhibitor of the present invention dissolved in dimethyl sulfoxide (DMSO), water, or a mixture thereof so as to be 0.4 to 40 μg / ml in each well (A to E: A is IF LAB Ltd., Ukraine, B, and C were purchased from PHARMEKS, Russia, and D and E were purchased from SPECS, the Netherlands), or 10 μl of ESGRO was added, and cultured in a 37 ° C., 5% CO 2 incubator for 4 days. DMSO was brought into the medium at a final concentration of 0.1% or less. Further, only DMSO was added to the control well so that the final concentration was 0.1%. The structures of differentiation inhibitors A to E are shown in FIG.
[0054]
"Quantification of alkaline phosphatase"
The alkaline phosphatase activity of ES cells was determined using p-NITROPHENYL PHOSPHATE SOLUTION (Moss Inc., PRODUCT NO.NPPD-1000, USA, or SIGMA, A-3469, USA) (hereinafter, p-NPP) And quantified. The medium is aspirated and removed from each well of ES cells cultured for 4 days by the method described in “ES cell differentiation inhibition assay 1”, and the cells are washed once with 100 μl of phosphate buffered saline (PBS). , 100 μl of p-NPP was added to each well and allowed to stand at room temperature for 10 minutes. 12.5 μl of 8M sodium hydroxide solution was added to each well to stop the reaction. The absorbance at 405 nm (OD 405) and absorbance at 690 nm (OD 690) of the solution were measured with an absorptiometer (Molecular Devices, model: SPECTRA MAX190). D. 405-O. D. The value calculated at 690 was defined as alkaline phosphatase activity. A graph of the quantitative results is shown in FIG. The differentiation inhibitors of the present invention, compounds A to E, significantly amplified alkaline phosphatase activity compared to DMSO (0.1%) as a control. That is, it can be seen that the differentiation inhibitor of the present invention supported the culture of undifferentiated ES cells.
[0055]
[Example 2]
"ES cell differentiation inhibition assay 2"
The D3ES cells cultured in the “ES cell culture” were washed twice with PBS. A 0.25% trypsin solution (GIBCO BRL 15090-046) was added and incubated at 37 ° C. for 5 minutes to desorb colonies of undifferentiated D3ES cells from the feeder. 5 ml of ES cell medium was added, cell colonies were dispersed using a small-diameter pipette, transferred to a 50 ml sterile tube, and pelleted by centrifugation at 1000 rpm for about 5 minutes in a tabletop centrifuge (Tomy Seiko). The supernatant was removed and the cells were resuspended in 20 ml of fresh ES cell medium, seeded in a 15 cm diameter cell culture dish pre-coated with 0.1% aqueous gelatin and incubated at 37 ° C. for 20 minutes. After 20 minutes, the medium containing floating cells was collected with a pipette, seeded again in a cell culture dish having a diameter of 15 cm coated with 0.1% gelatin aqueous solution, and incubated at 37 ° C. for 20 minutes. After 20 minutes, the medium containing floating cells was collected with a pipette, transferred to a 50 ml sterilized tube, pelleted by centrifugation at 1000 rpm for about 5 minutes in a tabletop centrifuge, the supernatant was removed, and 5 ml of ES cell assay medium was obtained. Resuspended. 8 × 10 8 in a 10 cm diameter cell culture dish previously coated with 0.1% gelatin aqueous solution ⁴ Cells were seeded so that the ES cell assay medium was 10 ml. To each dish, 1 ml of dimethyl sulfoxide (DMSO), the differentiation inhibitor of the present invention (A to E) or ESGRO dissolved in a medium or a mixture thereof so as to be 40 μg / ml, is added at 37 ° C., 5 ° C. % CO ₂ The cells were cultured for 7 days in an incubator. DMSO was brought into the medium at a final concentration of 0.1% or less.
[0056]
“Evaluation of SSEA-1 antigen expression level”
The cells cultured in the “ES cell differentiation inhibition assay 2” were washed twice with PBS, and the cells were detached from the dish with a cell scraper. 6 × 10 cells ⁵ 300 μl of Hanks balanced salt solution (HBSS, manufactured by Invitrogen) containing 2% FBS was added so as to form an individual. 30 μl of MX-SSEA-1 antibody (manufactured by Kyowa Medex) diluted 50-fold with HBSS was added and allowed to stand on ice for 40 minutes. After washing twice with HBSS (1 ml), it was redispersed in HBSS (300 μl), added with 30 μl of 20-fold FITC-Goat anti Mouse IgM antibody (manufactured by ZYMED) with HBSS, and kept on ice for 30 minutes while protected from light. After washing twice with HBSS (1 ml), it was redispersed in HBSS (1 ml), and 100 μl of 20 μg / ml PI solution (manufactured by Dojindo) was added to obtain a sample used for flow cytometer measurement. The sample used for flow cytometer measurement was measured after passing through a nylon mesh having a pore diameter of 100 μm for the purpose of removing aggregates. FACSCalibur (BECTON Dickinson, USA) was used as the flow cytometer, and CELLQuest (BECTON Dickinson, USA) was used as the measurement condition data collection and analysis software. The results are shown in FIG. The average fluorescence intensity per cell of cells cultured with each differentiation inhibitor added, that is, the expression intensity of SSEA-1 antigen per cell is significantly higher than that of cells cultured in a medium containing no differentiation inhibitor. It was enhanced. Moreover, when the same experiment was conducted about the compound B￣E, it was similarly shown that the expression level of SSEA-1 was significantly high. That is, it was revealed that the differentiation inhibitor of the present invention maintains undifferentiated ES cells.
[0057]
[Example 3]
"STAT3 activation assay"
The D3ES cells cultured in the “ES cell culture” are washed twice with PBS. A 0.25% trypsin solution (GIBCO BRL) is added and incubated at 37 ° C. for 5 minutes to desorb undifferentiated D3ES cell colonies from the feeder. 5 ml of ES cell medium was added, cell colonies were dispersed using a small-diameter pipette, transferred to a 15 ml sterilized tube, and pelleted by centrifugation at 800 rpm for about 5 minutes in a tabletop centrifuge (Tomy Seiko). The supernatant was removed and the cells were resuspended in 5 ml of fresh ES cell medium, seeded in a 10 cm diameter cell culture dish pre-coated with 0.1% aqueous gelatin and incubated at 37 ° C. for 20 minutes. After 20 minutes, the medium containing floating cells was collected with a pipette, transferred to a 15 ml sterilized tube, pelleted by centrifugation at 800 rpm for about 5 minutes in a tabletop centrifuge, the supernatant was removed, and 5 ml of ES cell assay medium was obtained. Resuspend.
[0058]
In a 24-well cell culture dish (FALCON Cat. No. 3047, USA) pre-coated with 0.1% gelatin aqueous solution, 1 × 10 5 per well ⁵ Cells were seeded at 500 μl of ES cell medium per well and incubated at 37 ° C., 5% CO 2. ₂ The cells were cultured for 12 to 24 hours in an incubator. Next, using LipofectAMINE 2000 (Invitrogen, USA), according to the attached protocol, pSTAT3-TA-Luc or pTA-Luc vector (Clontech, USA) was added to ES cells at 0.9 μg per well. Transfected. Further, as an internal control, 0.1 μg of each well was simultaneously transfected with pRL-TK vector (Promega, USA). After culturing at 37 ° C. in a 5% CO 2 incubator for 4 hours, the medium was aspirated and washed twice with PBS, and then 500 μl of ES assay medium was added to each well. Next, a differentiation inhibitor dissolved in dimethyl sulfoxide (DMSO) or water or a mixture thereof so as to be 0.4 to 40 μg / ml in each well, or ES assay medium so as to be 10 to 10,000 units / ml. 50 μl of ESGRO diluted in 1 was added and cultured at 37 ° C. in a 5% CO 2 incubator for 6-24 hours. Subsequently, using Picker Gene Dual Sea Pansy (manufactured by Toyo Ink Manufacturing Co., Ltd., Japan), the luminescence signal of each cell by the luciferase reporter enzyme was measured according to the attached protocol. As shown in FIG. 4, ESGRRO increased the expression of the reporter enzyme by pSTAT3-TA-Luc in a dose-dependent manner, whereas Compound B of the present invention did not induce the expression of the reporter enzyme.
Moreover, although the same experiment was done about compound A and CE, the expression of reporter enzyme was not induced | guided | derived similarly to the time of using compound B.
[0059]
[Brief description of the drawings]
FIG. 1 shows the structures of compounds A to E of the present invention.
Compounds A to E are R of formula (I) ¹ ~ R ⁹ Are represented by the groups or elements shown in the table below.
FIG. 2 shows the results of quantification of alkaline phosphatase of the present invention.
FIG. 3 shows the results of average fluorescence intensity in evaluation of the expression level of SSEA-1 antigen of the present invention.
FIG. 4 shows the results of the STAT3 activation assay of the present invention.

Claims (11)

A stem cell differentiation inhibitor comprising a low molecular compound as an active ingredient. The differentiation inhibitor according to claim 1, wherein the low molecular compound is an indole derivative. The differentiation inhibitor according to claim 2, wherein the indole derivative is a compound represented by the general formula (I), a salt thereof or an ester compound.

(Wherein R ^{1 to} R ⁹ represent the same or different electron-withdrawing groups, electron-donating groups, or hydrogen atoms.) The differentiation inhibitor according to claim 3, wherein R ^{1 to} R ⁹ are a group or an atom selected from the group consisting of an alkyl group, an alkoxy group, a hydroxyl group, a nitro group, an arylaminovinyl group, and a hydrogen atom. The differentiation inhibitor according to claim 2, wherein the indole derivative is a compound represented by the general formula (II), a salt thereof or an ester compound.

A ^{1 to} A ³ represent a lower alkyl group, a lower alkoxy group, or a hydrogen atom, which may be the same, and A ⁴ represents a lower alkyl group or an arylaminovinyl group. The compound represented by formula (I) is:
2-methyl-3-nitro-1-phenyl-1H-indole-6-ol, 1- (4-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol, 2-methyl- 3-nitro-1-p-tolyl-1H-indole-6-ol, 2- [2- (4-methoxy-phenylamino) -vinyl] -3-nitro-1-p-tolyl-1H-indole-6 The differentiation suppression according to claim 2, which is a compound of any one of -ol or 1- (2-methoxy-phenyl) -2-methyl-3-nitro-1H-indole-6-ol, a salt thereof or an ester compound thereof. Agent. The differentiation inhibitor according to any one of claims 1 to 6, wherein the low molecular compound is a compound that does not activate STAT3 (signal transducer and activator of transcription 3). A stem cell culture method comprising culturing stem cells in an undifferentiated state using the differentiation inhibitor according to any one of claims 1 to 7. An embryonic stem cell culture method comprising culturing embryonic stem cells in an undifferentiated state using the differentiation inhibitor according to any one of claims 1 to 8. A culture solution for proliferating stem cells in an undifferentiated state, containing the differentiation inhibitor according to any one of claims 1 to 7 as an active ingredient. Stem cells cultured in an undifferentiated state using the differentiation inhibitor according to any one of claims 1 to 7.

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