JP2017029153A - 分娩後の哺乳動物の胎盤、その使用およびそれに由来する胎盤幹細胞 - Google Patents
分娩後の哺乳動物の胎盤、その使用およびそれに由来する胎盤幹細胞 Download PDFInfo
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Abstract
【解決手段】瀉血した胎盤を、好ましくは抗凝固剤溶液を用いて、灌流して残留細胞を洗い流すことにより胎盤を処理して、残留臍帯血を除去する。瀉血した胎盤からの残留細胞および灌流液を収集した後、この残留細胞および灌流液から胚様幹細胞を分離する。
【選択図】なし
Description
本発明は、子宮からの娩出後、例えば出産後、の胎盤を瀉血および灌流する方法に関する。本発明は、胎盤および外因性の供給源に由来する胚様幹細胞を増殖するために、分離した胎盤を処理および培養する方法に関する。本発明はさらに、生物学的物質または培養細胞、組織および類器官を生産するバイオリアクターとして、培養した胎盤を使用することに関する。本発明はまた、幹細胞の収集および増殖、特に、胎盤からの胚様幹細胞およびその他の多能性幹細胞の収集にも関する。本発明は、分娩後の胎盤に由来する胚様幹細胞に関する。
ヒト幹細胞の同定、分離および生産には相当の関心が集まっている。ヒト幹細胞は、多様な成熟ヒト細胞系統を発生させる能力がある全能性または多分化能性の前駆細胞である。この能力は、器官および組織発生に必要な細胞分化および専門化のための基礎として働く。
本発明は、子宮からの娩出後に、多能性幹細胞(例えば、前駆細胞)、胚様幹細胞およびその他の生物学的物質をもたらすように処理され培養された哺乳動物(好ましくはヒト)の胎盤に関する。特に、本発明は、分娩後の胎盤を灌流および瀉血する方法を提供する。本発明は、子宮からの胎盤の娩出後、少なくとも2時間から48時間以上にわたり、無菌条件下で該胎盤を瀉血および灌流する方法を提供する。好ましい実施形態では、抗凝血因子のように、瀉血を高める因子を含む溶液を用いて、胎盤を灌流する。別の実施形態では、抗菌および抗ウイルス薬のように、無菌条件を高める因子を含む溶液を用いて、胎盤を灌流する。好ましい実施形態では、増殖因子を含む溶液で胎盤を灌流する。増殖因子およびその他の培養成分を含むが、抗凝血剤は含まない溶液を培養溶液と呼ぶ。
本明細書で用いる「バイオリアクター」という用語は、細胞を増殖する、生物学的物質を生産または発現する、ならびに、細胞、組織、類臓器、ウイルス、タンパク質、ポリヌクレオチドおよび微生物を生育または培養するためのex vivoシステムを意味する。
本発明者は、思いがけなく、分娩後の胎盤が、分娩後適切に処理すれば、活性化することができる静止細胞を含むことをみいだした。例えば、子宮からの娩出後、胎盤を出来るだけ迅速に瀉血すると、アポトーシスが阻止されるか、または最小限に抑えられる。次に、瀉血後できるだけ早期に、胎盤を灌流して、器官に存在する血液、残留細胞、タンパク質、因子およびその他の材料を除去する。物質残屑も胎盤から除去することができる。灌流は、通常、少なくとも2〜24時間以上にわたり適当な灌流液で継続する。いくつかの別の実施形態では、胎盤は、少なくとも4、6、8、10、12、14、16、18、20および22時間灌流する。換言すれば、本発明は、瀉血および灌流を十分な時間実施することにより、分娩後の胎盤の細胞を活性化できるという発見に少なくとも部分的に基づいている。従って、胚様幹細胞の豊富な供給源として胎盤を容易に用いることができ、これらの胚様幹細胞は、薬物の発見、疾患の治療および予防、特に、移植手術または移植療法、ならびに、前駆細胞、組織および類臓器の形成を含めて、様々な研究に用いることができる。
5.1.1. 胎盤の前処理
本発明の方法によれば、出産後、娩出直後のヒト胎盤を回収し、特定の実施形態では、胎盤中の臍帯血を回収する。特定の実施形態では、この胎盤を通常の臍帯血回収方法に付す。このような臍帯血回収は、例えば、LifeBank Inc., Cedar Knolls, N,J., ViaCord, Cord Blood Registry and Cryocellから商業的に入手することができる。臍帯血は、胎盤の娩出直後に排液することができる。
本発明は、瀉血した、すなわち、分娩および/または通常の臍帯血回収法の後に残った臍帯血を完全に排出した胎盤から、限定するものではないが、胚様幹細胞などの幹または前駆細胞を回収する方法を提供する。本発明の方法によれば、胎盤は、瀉血すると共に、抗凝固剤(例えば、ヘパリン、ワルファリンナトリウム)を溶解させた水性等張液などの好適な水性灌流液で灌流する。このような灌流用の水性等張液は当業者には公知であり、例えば、0.9 N塩化ナトリウム溶液が挙げられる。灌流液は、残留臍帯血の血餅形成を防止するのに十分な濃度で、ヘパリンまたはワルファリンナトリウムのような抗凝固剤を含むのが好ましい。特定の実施形態では、1〜100単位のヘパリン濃度を使用し、好ましくは、1ml当たり1〜10単位のヘパリン濃度を用いる。一実施形態では、瀉血中、および瀉血直後に、アポトーシス阻害剤、例えば、ラジカルスカベンジャー、特に酸素遊離基スカベンジャーを用いてから、これらの薬剤を胎盤から洗い流す。本発明のこの実施形態によれば、単離された胎盤を低温条件下で保存することにより、アポトーシスを防止または阻害する。
胎盤の瀉血および十分な時間の灌流後、瀉血および灌流した胎盤の微小循環へ胚様幹細胞が移動しているのが観察されるが、この微小循環において、本発明の方法に従い、好ましくは灌流で収集容器中に洗い流すことにより、上記胚様幹細胞を収集する。単離された胎盤の灌流は、残留臍帯血を除去するのに役立つだけではなく、酸素を含む好適な栄養分を胎盤に供給するにも有用である。この胎盤を培養し、残留臍帯血細胞の除去に用いたものと同様の溶液(好ましくは、抗凝固剤を添加せずに)を用いて、灌流することができる。
前述したように、胎盤の瀉血および灌流後、胚様幹細胞は排液された空の微小循環に移動し、この微小循環において、好ましくは収集容器中に流出灌流液を収集することにより、胚様幹細胞を本発明に従い収集する。
本発明の方法に従って取得された胚様幹細胞には、多分化能性細胞、すなわち、完全な分化能を有し、自己複製能があり、かつ組織内で休眠または静止状態のままでいられる細胞が含まれる。胎盤から取得できる幹細胞には、胚様幹細胞、多能性細胞、前駆細胞、および繊維芽細胞様細胞が含まれる。
瀉血および/または培養した胎盤細胞を細胞、組織および器官の培養用バイオリアクターとして用いることができる。胎盤中胚葉は、器官発生および組織新生に必要な、小分子および増殖因子、リポ多糖類、および細胞外マトリックスタンパク質を豊富に含む理想的なストロマ環境を提供する。
本発明の胚様幹細胞は、非常に多様な治療プロトコルに用いることができ、これらのプロトコルでは、幹細胞または前駆細胞集団のような所望の細胞集団の植付け(engraftment)、移植もしくは注入により、身体の組織または器官が増大、修復または置換される。本発明の胚様幹細胞を用いて、既存の組織を置換または増大したり、新しいまたは改変した組織を導入したり、あるいは、生物学的組織または構造を一緒に結合したりすることができる。また、本発明の胚様幹細胞は、一般に胚性幹細胞を用いる治療プロトコルにおいて、胚性幹細胞の代わりに用いることができる。
本発明は、条件付けまたは非条件付けヒト前駆幹細胞の移植前または後に、1回または複数回投与して、胎盤由来のヒト多分化能性および多能性前駆幹細胞が中胚葉および/または造血系列細胞に分化するのを阻害し、モジュレートし、かつ/または調節するのに十分な効果を及ぼす、有効な1回量および/または複数回量を含む医薬組成物を包含する。
6.1. 実施例1:排液した胎盤の灌流液から回収した細胞型の分析
この実施例では、本発明の方法により培養した胎盤の流出灌流液から回収された細胞型の分析について説明する。
以下の実施例は、本発明の方法に従い、胎盤の灌流およびインキュベーションにより取得された細胞の分析について説明する。
胎盤ドナーは、民間の臍帯血バンクプログラムに登録し、臍帯血の回収後瀉血した胎盤の研究目的での使用を認めるインフォームドコンセントを提供した妊娠中の母親から募った。ドナーデータは内密である。これらのドナーは、低温保存のための臍帯血サンプルの通常処理から得られたデータの使用も認めた。これにより、以下に記載する実験方法を用いて、収集した臍帯血と、回収した流出灌流液の組成を比較することができた。
分娩から12〜24時間以内にドナーの胎盤を室温で処理した。処理前に、膜を除去し、母親の部位(maternal site)を洗い流して残留血液を除いた。臍帯血管に、血液サンプル収集に用いられる20ゲージのバタフライ(Butterfly)針からなるカテーテルを挿入した。
室温で5,000 x gの遠心分離を15分実施することにより、灌流液から細胞を回収した。この手順は、混入している残屑および血小板から細胞を分離するのに役立った。細胞ペレットを2U/mlヘパリンおよび2mM EDTA(GibcoBRL, NY)を含有するIMDM無血清培地中で再懸濁させた。リンフォプレップ(Lymphoprep)(Nycomed Pharma、オスロ、ノルウェー)を用いて、製造業者の推奨手順に従い、全単核細胞画分を単離した後、単核細胞画分を再懸濁させた。血球計を用いて細胞を数えた。トリパンブルー排除により生存可能性を評価した。間葉細胞の単離は、0.2%EDTAを含む0.05%トリプシン溶液(Sigma、セントルイス、MO)を用いた「ディファレンシャルトリプシン処理」により達成された。ディファレンシャルトリプシン処理が可能であった理由は、繊維芽細胞様細胞がプラスチック表面から約5分以内に脱離したのに対して、他の付着集団は20〜30分のインキュベーションを必要としたためである。トリプシン処理ならびにトリプシン中和溶液(TNS, Bio Whittaker)を用いたトリプシン中和後に、脱離した繊維芽細胞様細胞を回収した。細胞をH.DMEM中で洗浄し、MSCGM中に再懸濁させた。
培養フラスコ中の付着細胞の顕微鏡検査により、形態が異なる細胞型が明らかになった。紡錘型の細胞、大きな核と多数の核周辺小液胞を有する丸い細胞、ならびに、数個の突起(そのうちの一つを介して星型細胞はフラスコに付着していた)のある星型の細胞がフラスコに付着しているのを観察した。これらの付着細胞のさらなる特性決定は試みなかったが、類似した細胞が骨髄、臍帯および末梢血液の培養物に認められたことから、非幹細胞様であると考えられた。最後にクラスターとして現れた繊維芽細胞様細胞は、MSC(間葉幹細胞)となる候補であり、ディファレンシャルトリプトシン処理により単離し、二次フラスコで継代培養した。トリプトシン処理後に、丸い細胞の位相差顕微鏡検査によって、細胞が高度に顆粒化していることがわかり、これは、研究室で作製したまたはBio Whittakerから購入した骨髄由来のMSCと見分けがつかなかった。継代培養すると、それらの初期相とは対照的に、数時間以内に付着した、胎盤由来の胚様幹細胞は特有の繊維芽細胞様の形状を呈し、基準の骨髄由来MSCと同じ増殖パターンを形成した。継代培養および再補給の間、さらに、緩く結合した単核細胞を洗浄して除去すると、この培養物は均一な状態のままで、繊維芽細胞様細胞以外の混入物が肉眼で一切認められなかった。
初期および後期画分から精製された単核細胞上のCD-34、CD-38、ならびに、その他の幹細胞関連表面マーカーの発現をフローサイトメトリーにより評価した。回収、選別した細胞をPBSにおいて洗浄してから、抗CD-34フィコエリトリンおよび抗CD38フルオレセインイソチオシアネート(Becton Dickinson、マウンテンビュー、CA)で二重染色した。
Claims (59)
- 無菌条件下で瀉血および灌流した、単離された哺乳動物胎盤。
- 胎盤を灌流するのに用いる溶液が抗凝固剤溶液を含む、請求項1に記載の単離された哺乳動物胎盤。
- 胎盤を灌流するのに用いる溶液が抗菌剤溶液を含む、請求項1に記載の単離された哺乳動物胎盤。
- 胎盤を灌流するのに用いる溶液が増殖因子を含む、請求項1に記載の単離された哺乳動物胎盤。
- 胎盤がヒト由来である、請求項1に記載の単離された哺乳動物胎盤。
- 子宮からの胎盤の娩出後約2〜24時間保存した、請求項1に記載の単離された哺乳動物胎盤。
- 胎盤からの胚様幹細胞および他の多能性幹細胞の生産を可能にする条件下で瀉血、灌流およびインキュベーションした、単離された哺乳動物胎盤。
- 約2〜24時間にわたってインキュベーションした、請求項7に記載の単離された哺乳動物胎盤。
- 約24〜48時間以上にわたって灌流またはインキュベーションした、請求項7に記載の単離された哺乳動物胎盤。
- 生存可能な胚様幹細胞を含む、単離され灌流された哺乳動物胎盤。
- 前記幹細胞がOCT-4-およびABC-p+である、請求項10に記載の単離された胎盤。
- 少なくとも2時間灌流した、請求項1に記載の単離された哺乳動物胎盤。
- 少なくとも11時間灌流した、請求項12に記載の単離された哺乳動物胎盤。
- 胎盤が分娩後に回収される、請求項10に記載の哺乳動物胎盤。
- 少なくとも24時間灌流した、請求項13に記載の単離された哺乳動物胎盤。
- 増殖因子を含む溶液で胎盤を灌流する、請求項10に記載の単離された哺乳動物胎盤。
- 増殖因子を含む溶液で灌流した、請求項7に記載の単離された哺乳動物胎盤。
- ヒト由来である、請求項7に記載の単離された哺乳動物胎盤。
- 子宮からの娩出後に胎盤を取得し、この胎盤を瀉血した後、無菌条件下で灌流することを含んでなる、哺乳動物胎盤の培養方法。
- 抗凝固剤溶液を含む溶液で胎盤を灌流する、請求項19に記載の方法。
- 抗菌剤を含む溶液で胎盤を灌流する、請求項19に記載の方法。
- 前記胎盤がヒト由来である、請求項19に記載の方法。
- 子宮からの娩出後約2〜24時間胎盤を保存する、請求項19に記載の方法。
- 前記娩出が分娩時である、請求項19に記載の方法。
- 胎盤を室温で保存する、請求項23に記載の方法。
- 前記胎盤を冷蔵または冷凍条件下で保存する、請求項23に記載の方法。
- 胎盤からの胚様幹細胞の生産を可能にする条件下で、この胎盤を培養することを含んでなる、瀉血および灌流した、単離された哺乳動物胎盤の培養方法。
- 胎盤を培養することがこの胎盤を灌流することを含む、請求項27に記載の方法。
- 約24〜48時間にわたって前記胎盤をインキュベートする、請求項27または28に記載の方法。
- 前記胎盤を少なくとも2時間灌流する、請求項27に記載の方法。
- 前記胎盤を少なくとも11時間灌流する、請求項30に記載の方法。
- 前記胎盤を少なくとも24時間灌流する、請求項31に記載の方法。
- 前記胎盤を48時間以上灌流する、請求項32に記載の方法。
- 前記胎盤を、増殖因子を含む溶液で灌流する、請求項28に記載の方法。
- 前記胎盤がヒト由来である、請求項27に記載の方法。
- OCT-4+およびABC-p+である、単離されたヒト胎盤幹細胞。
- 前記細胞がヒト細胞である、請求項36に記載の幹細胞。
- 少なくとも次の特徴:CD10+、CD29+、CD34-、CD44+、CD45-、CD54+、CD90+、SH2+、SH3+、SH4+、SSEA3-、SSEA4-、OCT-4+およびABC-p+を有する、単離された哺乳動物胎盤幹細胞。
- 前記細胞がSSEA3-およびSSEA4-である、請求項35に記載の単離された胎盤幹細胞。
- 分娩後のヒト胎盤を少なくとも11時間瀉血および灌流した後、この胎盤から単離したヒト胎盤幹細胞。
- (a)瀉血および灌流しておいた分娩後の哺乳動物胎盤;
(b)上記胎盤をインキュベートまたは培養する手段;および
(c)幹細胞を検出する手段;
を含んでなる幹細胞生産装置。 - 幹細胞を収集するための収集装置をさらに含む、請求項39に記載の装置。
- 培養条件をモニタリングおよび調節する手段をさらに含む、請求項39に記載の装置。
- 前記モニタリングおよび調節がコンピュータ化される、請求項41に記載の装置。
- 細胞分離装置をさらに含む、請求項39に記載の装置。
- 請求項35、37または38に記載のヒト胎盤幹細胞を投与することを含んでなる、ヒト疾患の治療方法。
- 請求項35、37または38に記載のヒト胎盤幹細胞を、それを必要とする患者に投与することを含んでなる、幹細胞の移植方法。
- 請求項35、37または38に記載のヒト胎盤幹細胞を含んでなる、医薬組成物。
- 臍帯血または胎盤血をさらに含む、請求項46に記載の医薬組成物。
- 請求項35、37または38に記載のヒト胎盤幹細胞から分化した前駆細胞。
- すべての細胞型に分化することができるヒト胎盤幹細胞の単離された均一集団。
- 少なくとも次の細胞表面マーカーOCT-4+およびABC-p+を示す生存可能なヒト胎盤幹細胞の均一集団。
- 多能性であるヒト胎盤幹細胞の単離された均一集団。
- 胎児由来でも母親由来でもない細胞を含んでなる、単離された胎盤。
- 幹細胞が胎盤に由来する、請求項49または51に記載の幹細胞。
- CD34+およびCD38-である細胞が富化された造血幹細胞の集団を含んでなる、骨髄移植用の組成物。
- CD34+およびCD38+である細胞を有する臍帯血をさらに含む、請求項53に記載の組成物。
- CD34-およびCD38-である細胞が富化された造血幹細胞の集団を含む、骨髄移植用の組成物。
- CD34+およびCD38+である細胞を有する臍帯血をさらに含む、請求項54に記載の組成物。
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WO2018150945A1 (ja) | 2017-02-20 | 2018-08-23 | 株式会社フジミインコーポレーテッド | シリコン基板中間研磨用組成物およびシリコン基板研磨用組成物セット |
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