JP2008514230A - 霊長類胚性幹細胞の培養 - Google Patents
霊長類胚性幹細胞の培養 Download PDFInfo
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Abstract
Description
決定済み。
(発明の背景)
本発明は、霊長類胚性幹細胞培養物の培養方法及びその方法で有用な培養液に関する。
ある側面において、本発明は、霊長類胚性幹細胞の培養方法を提供する。幹細胞を、本質的に哺乳類胎児血清を含まず(好ましくは、本質的にどのような動物血清をも含まず)繊維芽細胞の支持細胞層以外の出所から供給される繊維芽細胞増殖因子を含む培養液中で培養する。ある好ましい方法においては、従来は幹細胞培養を維持するために必要であった繊維芽細胞の支持細胞層は、十分量の繊維芽細胞増殖因子の添加により不要なものとなる。
幾つかの以下の実験において、発明者の一人は、培養液への血清添加なしでヒトES細胞株を培養する本発明の方法及び培養液系を使用した。2つのクローン化的に得られたヒトES細胞株は、成分として血清を含まない培養液中で培養された時に、クローン的な派生の後8ヶ月以上も増殖し、3つの胚体胚葉すべての後生的派生体への分化能を保持していた。
以下の追加的実験は、FGFを高濃度で含むが血清及び支持細胞の存在しない状態におけるES細胞株の培養に向けられた。3つの異なる培養液配合物が、この研究において用いられたが、これらの培養液配合物とは、ここでは、UM100、BM+及びDHEMのことである。専門語UM100は、100 ng/ml のbFGFが添加された非調製培養液のことである。このUM100培養液は、Gibco Knockout Serum Replacer 製品を含むが、あらゆる種類の繊維芽細胞の支持細胞の使用を含まず若しくは必要としない。BM+培養液は、基礎的な培養液(DMEM/F12)に添加物を加えたものであり、下記に記載されているように、これもまた支持細胞なしで細胞の培養を可能にするが、この培養液は血清代替物製品を除いている。最後に、DHEMという名前は、確定したヒト胚性幹細胞培養液のことである。この培養液もまた、下記に記載されているように、ウシアルブミンを含むBM+培養液とは対照的に、完全に無機成分及びヒト蛋白だけを含む状態でヒトES細胞の培養、クローニング及び永久増殖に十分である。
UM100培養液は、次のとおり調製された。80%(v/v)DMEM/F12(Gibco/Invitrogen)並びに1 mM グルタミン(Gibco/Invitrogen)、0.1 mM β-メルカプトエタノール(Sigma-St.Louis, MO)及び1 % 非必須アミノ酸ストック(Gibco/Invitrogen)を補完した20%(v/v)Knockout-Serum Replacer(Gibco/Invitrogen)からなる非調製培養液(UM)。この培養液を完成させるために、100 ng/ml のbFGFが添加され、培養液は、0.22 μM ナイロンフィルター(Nalgene)を通して濾過された。
各種の培養液中でのヒトES細胞の増殖速度を測定するために、細胞はおよそ5x105 cells/well の細胞密度で6ウェル組織培養プレート(Nalgene)に三つ揃いで撒かれた。3日目、5日目、7日目に、三つ揃いのウェルは、トリプシン/EDTA(Gibco/Invitrogen)で処理され、個別化され、細胞数が測定された。7日目に、追加的なウェルが、トリプシンで処理され、細胞数が測定され、およそ2x105 cells/well の細胞密度で新しいプレートに撒きなおすために使用された。トリプシン処理されてきた7日目の培養は、ES細胞の表面マーカーであるOct4, SSEA4及びTral-60がFACS分析により定量分析された。増殖速度は、3つの継続的な継代に対して収集された。増殖速度の実験は、UM100で培養されたヒトES細胞が、CM(調製培地)で培養されたヒトES細胞と同程度にしっかりと育つことを示唆している。
各種の培養液中でのヒトES細胞の接着率を測定するために、細胞は2x105 cells/well の細胞密度で6ウェル組織培養プレート(Nalgene)に撒かれた。30分から48時間までの範囲の時点において、接着していない細胞は洗い流され、接着している細胞はトリプシン/EDTA(Gibco/Invitrogen)で剥がされ、数が測定された。これらの実験は、UM100の増殖速度のデータが、UM100とCMに対する同等の増殖速度ではなく、より優れた細胞接着とより緩やかな増殖の組み合わせによるものであるのかどうかを調べるために行われた。我々は、接着の割合が、試験が行われた全ての時点において両方の培養液に対して同等であることを発見した。このように、どちらの細胞も同じ速度で増殖した。
ヒトES細胞は、トリプシン/EDTA(Gibco/Invitrogen)+ 2 % ヒヨコ血清(ICN Biomedicals, Inc., Aurora, OH)を37℃で10分間処理して6ウェル組織培養プレート(Nalgene)から剥がされた。細胞は、等容量のFACS Buffer(PBS + 2 % FBS + 0.1 % アジ化ナトリウム)に希釈され、80 μMの細胞濾過器(Nalgene)を通して濾過された。ペレットは、1000 RPMで5分間の遠心分離により集められ、1 ml の0.5 % パラホルムアルデヒド中に再び懸濁させられた。ヒトES細胞は、37℃で10分間固定され、そのペレットは、上述の通り集められた。そのES細胞は、2 ml のFACS Bufferに再び懸濁させられ、総細胞数が血球計で測定された。細胞は、上述の通りペレット化され、90 % エタノール中に氷上で30分間浸透化された。ヒトES細胞は、上述の通りペレット化され、1x105 cellsの細胞が、FACSチューブ(Becton Dickinson)中の1 ml のFACS Buffer + 0.1 % Triton X-100(Sigma)に希釈された。hESCが上述の通りペレット化され、FACS Buffer + 0.1 % Triton X-100(Sigma)に希釈された50 μlの1次抗体中で再び懸濁された。適切な対照抗体のサンプルが、並行して用いられた。hESCは、4℃で一晩中インキュベートされた。上清は流し除かれ、細胞は、50 μlの二次抗体(Molecular Probes/Invitrogen)中に室温において暗がりで30分間インキュベートされた。FACS分析は、CellQuest Software(Becton Dickinson)を用いてFascalibur(Becton Dickinson)セルソーターで行われた。FACS分析を行うためのこの方法は、このようにES細胞を持っていることを示すために細胞表面のマーカーを検出することを可能にする。観察されたその結果は、UM100中で培養されたヒトES細胞が個体数としてOct-4に対して90 % 陽性であるということであった。これは、CMで培養されたES細胞と比べても遜色なく、その細胞がES細胞集団であることを確かなものとしている。SSEA4及びTral-60の分析に対しては、その方法は、細胞がパラホルムアルデヒド若しくはメタノール中で取り扱われないことを除けば、Oct-4に対してのものと同様に行われた。細胞染色の後に、細胞は、FACS緩衝液(Tritonを含まない)中に再び懸濁させられ、上述の通りFACS緩衝液(繰り返しになるが、Tritonを含まない)中で適切な抗体と共に分析された。未分化ES細胞培養は、これらの2つの細胞表面マーカーに対して同様に平均しておよそ90 % 陽性であった。これは、上記で議論されたFACS分析によって実証された。
ヒトES細胞株H1細胞は、ここでは、ヒトES細胞の形態及び特徴を保持したままUM100培養液中で33継代以上(164回の細胞数倍増以上)培養された。H1細胞は、ヒトES細胞の形態及び特徴を保持したままBM+培養液中で6継代以上(70日間)培養された。H9細胞は、DHEM培養液中で5継代以上(67日間)培養された。H9及びH7ヒトES細胞もまた、UM100培養液中で未分化状態のままそれぞれ22継代及び21継代培養された。BM+及びUM100で培養された細胞のその後の試験は、正常の核型であることを証明した。
ヒトES細胞の株H1は、調製培養液中の標準的な条件下で試験培養液に移される前に3回継代の間培養された。その試験条件に対して、細胞は24時間(day 0)調整培養液上で培養され、それから翌日(day 1)、試験培養液に移された。その後、細胞は、それぞれの試験培養液中で培養された。ヒトES細胞株H9は、また、並行して試験培養液に移される前に調整培養液中のマトリゲル上で5継代の間培養された。
本発明は、霊長類胚性幹細胞の培養方法及びそれと共に用いる培養液を提供する。
Claims (11)
- ヒト胚性幹細胞の培養方法であって:
幹細胞を本質的に哺乳類胎児血清を含まない培養液中で培養し、アミノ酸類、ビタミン類、塩類、無機物類、トランスフェリン若しくはトランスフェリン代替物、インスリン若しくはインスリン代替物、アルブミン及び支持細胞層以外の出所から供給され少なくとも維持濃度で存在する繊維芽細胞増殖因子を含む幹細胞培養液中で培養することを含み、前記培養液は、支持細胞層若しくは支持細胞層への培養液の暴露を必要としない少なくとも6回継代されたヒト未分化増殖型正倍数性胚性幹細胞の培養と増殖を支持する能力のある培養方法。 - 培養液中に本質的にいずれの動物の血清をも含まない請求項1に記載の方法。
- FGFが、FGF2、FGF4、FGF9、FGF17及びFGF18からなる群より選択された請求項1に記載の方法。
- FGFが、100 ng/mlで培養液中に存在するFGF2である請求項1に記載の方法。
- 血清及び支持細胞層を含まない既知組成培養液中でのヒト胚性幹細胞の培養方法であって:
幹細胞をアルブミン、アミノ酸類、ビタミン類、無機物類、少なくとも1つのトランスフェリン若しくはトランスフェリン代替物、少なくとも1つのインスリン若しくはインスリン代替物を含む培養液で培養し、前記培養液は本質的に哺乳類胎児血清を含まず、繊維芽細胞増殖因子シグナル伝達受容体を活性化する能力のある少なくともおよそ100 ng/mlの繊維芽細胞増殖因子を含み、前記繊維芽細胞増殖因子は支持細胞層以外の出所から供給され、前記培養液は支持細胞層若しくは調製培養液を含まないで幹細胞が未分化状態で増殖することを支持する培養方法。 - 前記の培養工程が、内胚葉、中胚葉及び外胚葉組織の派生体へ分化する幹細胞の能力を維持し、幹細胞の核型を維持したままの一ヶ月間以上の培養液中での胚性幹細胞の増殖を含む請求項5に記載の方法。
- FGFが、FGF2、FGF4、FGF9、FGF17及びFGF18からなる群より選択された請求項5に記載の方法。
- ヒト胚性幹細胞の培養物であって:
ヒト胚性幹細胞を含み;
アルブミン、アミノ酸類、ビタミン類、少なくとも1つのトランスフェリン若しくはトランスフェリン代替物、少なくとも1つのインスリン若しくはインスリン代替物を含む幹細胞培養液であって、本質的に哺乳類胎児血清を含まず、少なくとも繊維芽細胞増殖因子シグナル伝達受容体を活性化する能力を有する維持濃度の繊維芽細胞増殖因子を含み、血清非存在下、支持細胞層非存在下及び支持細胞層に暴露された培養液も非存在下で永久に幹細胞を培養可能な培養液を含み、
幹細胞が未分化状態において永久に正常な核型状態であることを維持することが可能な培養物。 - 繊維芽細胞増殖因子が、少なくともおよそ100 ng/ml の濃度で培養液中に存在するFGF2である請求項8に記載の培養物。
- 支持細胞層非依存的なヒト胚性幹細胞の培養物であって:
幹細胞培養液中のヒト胚性幹細胞を含み;
前記幹細胞培養液は、アルブミン、アミノ酸類、ビタミン類、無機物類、少なくとも1つのトランスフェリン若しくはトランスフェリン代替物、少なくとも1つのインスリン若しくはインスリン代替物を含み、本質的に哺乳類胎児血清を含まず、FGF2、FGF4、FGF9、FGF17及びFGF18からなる群より選択された繊維芽細胞増殖因子を少なくとも維持濃度で含み;
幹細胞が正倍数体及び未分化状態であり続ける支持細胞非依存的な培養物。 - 繊維芽細胞増殖因子が、少なくともおよそ100 ng/ml の濃度で存在する請求項10に記載の培養物。
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CN101072868A (zh) | 2007-11-14 |
CA2582566A1 (en) | 2006-04-06 |
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CA2582566C (en) | 2014-09-09 |
EP1799811B1 (en) | 2020-03-25 |
KR20070060135A (ko) | 2007-06-12 |
WO2006036925A1 (en) | 2006-04-06 |
AU2005289597B2 (en) | 2011-06-09 |
AU2005289597A1 (en) | 2006-04-06 |
GB2433943B (en) | 2010-06-16 |
EP1799811A1 (en) | 2007-06-27 |
CN101072868B (zh) | 2015-04-15 |
US20090023208A1 (en) | 2009-01-22 |
GB2433943A (en) | 2007-07-11 |
GB0707395D0 (en) | 2007-05-23 |
US7439064B2 (en) | 2008-10-21 |
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