JP2010533488A - 胚性幹細胞をaqp−1発現細胞へ分化させるための方法 - Google Patents
胚性幹細胞をaqp−1発現細胞へ分化させるための方法 Download PDFInfo
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Abstract
Description
本願は、2007年7月19日に出願された、米国仮特許出願第60/929,960号の恩典およびそれからの優先権を主張し、その内容は、参照により本明細書に完全に組み込まれる。
本発明は、胚性幹細胞をAQP-1発現細胞へ分化させるための方法に関する。
器官の失われた機能の交換を目指す再生医療における種々のアプローチは、十分な細胞の供給源の不足によって制限されている。従って、かなりの研究が、可能性のある細胞供給源としてヒト胚性幹(hES)細胞を探究することに注がれてきた。ヒト胚性幹(hES)細胞は、無限に自己再生することができ、多能性であり;即ち、これらの細胞は、それらが由来する生物においてみられる事実上あらゆるタイプの細胞へ分化することができる(2、3(非特許文献1、2))。従って、hES細胞は、種々の研究および医療目的における使用について種々の細胞型の供給物を提供し得る。
造血細胞、神経細胞および心臓細胞へのhES細胞のインビトロ分化が観察され(1、13、14)、研究によって、異なる成長因子の存在を含む異なる培養条件が、特定の細胞型の形成および成長に好都合であることが示された(13、15、16)。しかし、ヒト腎上皮細胞へのhES細胞の指向性インビトロ分化の報告はなかった。
実施例1
この実施例では、ヒト胚性幹(hES)を、異なる細胞外基質(ECM)成分を有する種々の培養表面上に播種し、腎上皮成長培地(REGM)中で培養した。この方法を、hESを腎上皮様細胞へ分化させるために適用した。REGM中において10日間培養した後、免疫組織化学およびウエスタンブロッティングによって、上皮表現型を示すAQP-1およびCK-18の発現が明らかになった。フローサイトメトリーによって、分化細胞は、ヒト腎近位尿細管中において見られるものと同様のCD受容体発現プロファイルを有することが示された。さらに、分化細胞は、機能的水輸送を示し、AQP-1発現を失うことなく灌流バイオリアクター中で7日間培養することができた。従って、この方法は、失われた腎機能を置き換えるための戦略において潜在的に使用され得る機能的腎上皮様細胞を作製するための分化プロトコルを提供する。
hES細胞培養および分化:HUES-7細胞株(Howard Hughes Medical Institute, USAから得た)を、20%の血清代替物(Invitrogen、カタログ番号10828028)、1%のGlutaMax(Invitrogen、カタログ番号35050061)、1%の非必須アミノ酸溶液(Invitrogen、カタログ番号11140-050)、1%のペニシリン-ストレプトマイシン(Invitrogen、カタログ番号15070-063)、0.004%の塩基性線維芽細胞成長因子(bFGF)(Invitrogen、カタログ番号13526029)、および0.1%の2-メルカプトエタノール(Invitrogen、カタログ番号21985023)を補ったノックアウトDMEM(Invitrogen, USA、カタログ番号10829018)中、ネオマイシン耐性初代マウス線維芽細胞、FVB株(カタログ番号PMEF-N, CHEMICON International, USA)上において、37℃、5%のCO2で培養した。初代マウス線維芽細胞を、ゼラチン0.1%(Sigma, USA)でコーティングされたT75フラスコ上に平板培養した。培養培地を毎日交換し、7〜10日間培養した。MEFの培養培地由来の馴化培養培地を回収し、10 ng/mlのbFGFと共にhES細胞で使用した。線維芽細胞層上で培養したhES細胞を、トリプシン処理し(0.05%のトリプシン、Invitrogen, USA)、かき集め、100-μmメッシュで濾過した。濾過した細胞を、MEFの培養培地由来の馴化培養培地を含有する、マトリゲル(商標)マトリックス(BD Biosciences, Germany)でコーティングされた(ノックアウトDMEM中に1:20希釈)培養フラスコ上で培養した。hES細胞を2回継代培養し、線維芽細胞を含まない培養条件を得た(3、18、21)。RT-PCR分析(図1c)によって検出された、Oct3/4(図1b)、Sox2、CD9およびNanogの発現によって、hES細胞の未分化状態が確認された。
全てのプライマーは、5'→3'方向で示されている:PCR産物を、アガロースゲル上で分離し、臭化エチジウムを使用して視覚化した。
Howard Hughes Medical Institute(USA)から得たHUES-7細胞株を、マウス胚性線維芽細胞(MEF)フィーダー層上で、胚状態に維持した(図1a)(16)。MEFフィーダー層から回収した馴化培地を使用して、ノックアウトダルベッコ改変イーグル培地(DMEM)中に希釈されたマトリゲル(商標)でコーティングされた表面上に、hES細胞を2回継代培養し(図1aおよびb)、線維芽細胞を含まない培養条件を得た(17〜19)。Oct3/4(図1b)、Sox2、CD9およびNanog(図1c)の発現によって、両方の条件下での未分化状態が確認された。
Claims (16)
- ヒト胚性幹(hES)細胞をAQP-1発現細胞へ分化させる方法であって、
AQP-1発現細胞へのhES細胞の分化を誘導するのに十分な条件下で、細胞外基質(ECM)分子の存在下で腎特異的培養培地中において未分化hES細胞を培養する工程
を含む、方法。 - 腎特異的培養培地が、腎上皮基礎培地または腎上皮成長培地を含む、請求項1記載の方法。
- ECM分子が、フィブロネクチン、ラミニン、コラーゲンIVおよびマトリゲルマトリックスのうちの少なくとも1つを含む、請求項1または請求項2記載の方法。
- 腎特異的培養培地が上皮成長因子を含む、請求項1〜3のいずれか一項記載の方法。
- 前記培養する工程の前または間に腎特異的培養培地へ骨形成タンパク質2を添加する工程をさらに含む、請求項1〜4のいずれか一項記載の方法。
- 前記培養する工程の前または間に約2.5〜約10 ng/mlの濃度で骨形成タンパク質2を腎特異的培養培地へ添加する工程をさらに含む方法であって、培養する工程が7〜10日間細胞を成長させる工程を含み、腎特異的培養培地が腎上皮成長培地を含み、ECM分子がマトリゲルマトリックスを含み、かついったん分化すると、細胞がAQP-1と、CK-18、β-カテニン、CD-326およびCD-133のうちの少なくとも1つとを発現する、請求項1記載の方法。
- 腎特異的培養培地中でかつ細胞外基質分子の存在下でヒト胚性幹細胞の集団から直接分化した細胞の集団であって、これらの細胞の約2%〜約50%がAQP-1を発現する、細胞の集団。
- 請求項1〜6のいずれか一項記載の方法によって調製された、請求項7記載の細胞の集団。
- 内腔を有する中空コアファイバーを含むバイオ人工尿細管補助装置であって、内腔が細胞外基質(ECM)分子および請求項7または8記載の細胞の集団でコーティングされている、バイオ人工尿細管補助装置。
- 内腔を有する中空コアファイバーを含むバイオ人工尿細管補助装置を提供する工程、および内腔に請求項7または8記載の細胞の集団を播種する工程を含む、バイオ人工尿細管補助装置を調製する方法であって、細胞の集団を播種する前に、内腔が細胞外基質分子でコーティングされる、方法。
- 内腔が、フィブロネクチン、ラミニン、コラーゲンIVおよびマトリゲルマトリックスのうちの少なくとも1つを含む細胞外基質分子でコーティングされる、請求項10記載の方法。
- 細胞の集団が、内腔をコーティングするコンフルエントな単層へと培養される、請求項10または請求項11記載の方法。
- その必要がある被験体における腎臓関連障害を治療する方法であって、有効量の請求項7または8記載の細胞の集団を、被験体における、AQP-1発現細胞が必要とされる部位に移植する工程を含む、方法。
- 細胞の集団が、請求項9記載のバイオ人工尿細管補助装置内に提供される、請求項13記載の方法。
- 被験体における腎臓関連障害を治療する方法であって、請求項9記載のバイオ人工尿細管補助装置を、その必要がある被験体へ、外部から接続する工程を含む、方法。
- 腎臓関連障害が、腎不全、腎症、糖尿病性腎症、ネフローゼ、ブライト病、腎機能不全、糸球体炎、糸球体硬化症または腎炎を含む、請求項13〜15のいずれか一項記載の方法。
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- 2008-07-21 EP EP08779492.1A patent/EP2176402B1/en not_active Not-in-force
- 2008-07-21 CN CN200880107638A patent/CN101848993A/zh active Pending
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2013
- 2013-06-13 US US13/917,269 patent/US20130268060A1/en not_active Abandoned
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2014509843A (ja) * | 2011-02-07 | 2014-04-24 | ライフ テクノロジーズ コーポレーション | 感受性化合物を安定化するための組成物および方法 |
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EP2176402A4 (en) | 2010-08-18 |
CN101848993A (zh) | 2010-09-29 |
US8481316B2 (en) | 2013-07-09 |
EP2176402B1 (en) | 2016-06-01 |
US20130268060A1 (en) | 2013-10-10 |
US20110070280A1 (en) | 2011-03-24 |
WO2009011663A1 (en) | 2009-01-22 |
EP2176402A1 (en) | 2010-04-21 |
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