JP6728541B2 - 哺乳類多能性幹細胞由来の内胚葉細胞を産生するための改良された方法 - Google Patents
哺乳類多能性幹細胞由来の内胚葉細胞を産生するための改良された方法 Download PDFInfo
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- JP6728541B2 JP6728541B2 JP2018221950A JP2018221950A JP6728541B2 JP 6728541 B2 JP6728541 B2 JP 6728541B2 JP 2018221950 A JP2018221950 A JP 2018221950A JP 2018221950 A JP2018221950 A JP 2018221950A JP 6728541 B2 JP6728541 B2 JP 6728541B2
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Description
前記分化期の哺乳類多能性幹細胞をDNA脱メチル化剤に曝露することとを含む哺乳類多能性幹細胞由来のDE細胞を産生する方法を提供する。
本明細書で用いられるところの「多能性の」又は「多能性」は、3種の胚葉(内胚葉、中胚葉及び外胚葉)の全ての種類の特殊化した細胞を形成する可能性を指し;子に発達可能な完全な胚を形成する能力である「全能性の」又は「全能性」とは区別されなければならない。
全ての(上で定義した)hPS細胞が、本発明の開始材料として使用することができる。以下の例では、特に、胚体内胚葉は、mEFフィーダー細胞で、樹立された未分化ヒト胚性幹細胞(hESC)からインビトロで誘導され(ハインズら2004)、フィーダーを含まない条件下で、維持された。本実験で使用される細胞株は、限定されるものではないが、hES細胞株SA167、SA181、SA461(セラーティス アーベー,ヨーテボリ,スウェーデン)であり、それらはハインズら2004によって記載されるように増殖させることができる。これらの細胞株は、NIH幹細胞レジストリー、英国幹細胞バンク及び欧州hESCレジストリーに収載され、依頼に応じて入手可能である。
肝細胞様細胞は、以下の典型的な基本プロトコールA及びBを使用することにより、hPS細胞から誘導されてもよい。
未分化hPS細胞が分離され、新たに調製した0日目培地に、直接播種された。異なる培地が新たに調製され、0、1、2、3、4、5、7日目に添加された。前処理培地は、セレクティス アーベー(アルヴィド ヴァーグレーンス ベッカ 20、41356 ヨーテボリ、スウェーデン)から入手可能である。
0日目
前処理培地
CHIR99021 3μM
ROCK阻害剤 5μM
1日目
前処理培地
CHIR99021 3μM
2日目
RPMI 1640(+0.1%PEST、+1%グルタマックス)
B27(1x)
アクチビンA 50ng/ml
CHIR99021 3μM
LY294002 5μM
3日目
RPMI 1640(+0.1%PEST、+1%グルタマックス)
B27(1x)
アクチビンA 50ng/ml
LY294002 5μM
4〜7日目
RPMI 1640(+0.1%PEST、+1%グルタマックス)
B27(1x)
アクチビンA 50ng/ml
未分化hPS細胞が分離され、新たに調製した0日目培地に、直接播種された。異なる培地が新たに調製され、0、1、2、3、4、5、7日目に添加された。前処理培地は、セレクティス アーベー(アルヴィド ヴァーグレーンス ベッカ 20、41356 ヨーテボリ、スウェーデン)から入手可能である。
0日目
前処理培地
CHIR99021 3μM
ROCK阻害剤 5μM
1日目
RPMI 1640(+0.1%PEST、+1%グルタマックス)
B27(1x)
アクチビンA 50ng/ml
CHIR99021 3μM
LY294002 5μM
2日目
RPMI 1640(+0.1%PEST、+1%グルタマックス)
B27(1x)
アクチビンA 50ng/ml
LY294002 5μM
3〜7日目
RPMI 1640(+0.1%PEST、+1%グルタマックス)
B27(1x)
アクチビンA 50ng/ml
継代d7については、プロトコールAを参照されたい。
基本プロトコールAに続いて(hESC及びhiPSC由来の胚体内胚葉の両方で)、細胞が、プレ内胚葉段階の間の異なる時点で、異なる期間、例えば、プロトコールの2〜3、2〜4、3〜4及び4〜6日目に、10nM 5−アザ−2−デオキシシチジンで処理された(hESC−DE:5azadCなし n=4、5azadC d2−3 n=4、d3−4 n=1、d2−4 n=1、d4−6 n=1;hiPSC−DE:5azadCなし n=5、5azadC d2−3 n=5、d3−4 n=2、d2−4 n=1、d4−6 n=1;nは個々の実験の数である)。
図1A:
2〜3日目に10nMの5−アザ−2−デオキシシチジンで処理されたhESCに由来するDE(図1A2)は、未処理の対照DE(図1A1)と比較して、より均一であり、より顕著な細胞間接着を有する。対照DEでのより高いOct4の発現及びNanog mRNAの発現に従って(図1Dを比較)、対照DEでの未分化細胞の存在に留意されたい(図1A1)。2〜4、3〜4及び4〜6日に、100nMの5−アザ−2−デオキシシチジンで細胞を処理したときにも、同様の結果が得られた。1nMの5−アザ−2−デオキシシチジンは、効果がより低かった(データは示されない)。
2〜3日目に10nMの5−アザ−2−デオキシシチジンで処理したHiPSC由来のDE(図1B2)は、対照DE(図1B1)と比較して、よりコンフルエントであり、より顕著な細胞間接着を有する。2〜4、3〜4及び4〜6日に、100nMの5−アザ−2−デオキシシチジンで細胞を処理したときにも、同様の結果が得られた。1nMの5−アザ−2−デオキシシチジンは、効果がより低かった(データは示されない)。
2〜3日目に10nMの5−アザ−2−デオキシシチジンで処理したHiPSC由来のDEは、未処理の対照と比較して、7日目にはるかに少ないOct4−免疫陽性細胞を有し、すなわち、より少ない未分化細胞が残り、脱メチル化剤での処理後、DEはより均一である。
未処理の対照と比較して、2〜3、3〜4、2〜4及び4〜6日目に10nMの5azadCで処理したhESC及びhiPSC由来のDEでは、幹細胞マーカーOct4の発現は、はるかに低かった(図1D1)。5azadC処理hESC由来のDEでは、幹細胞マーカーNanogのmRNA発現が大幅に減少したのに対して、hiPSC由来のDEでは、幹細胞マーカーNanogのmRNA発現は主に影響を受けない(図1D1)。未処理の対照と比較して、5azadC処理hESC及びhiPSC由来のDEで、DEマーカーSox17、Cxcr4、FoxA2及びhHexの発現は増加し、効果はhiPSC由来のDEと比較してhESC由来のDEでより強い(図1D3〜6)。Sox7のmRNAレベルを増加させる2〜4及び4〜6日の5azadC処理を除き、対照及び5azadC処理hESC及びhiPSC由来のDEの両方で、胚体外マーカーSox7の発現は非常に低い。
基本プロトコールA又はBに続いて、27個のhPSC株由来の細胞は、胚体内胚葉へのhPSの分化期の2〜3日目に、10nMの5−アザ−2−デオキシシチジンで処理された(プロトコールA:ChiPSC14、ChiPSC19、ChiPSC22、P11015、SA167、SA181、SA461及びVal9;プロトコールB:ChiPSC4、ChiPSC6b、ChiPSC7、ChiPSC8、ChiPSC9、ChiPSC10、ChiPSC11、ChiPSC13、ChiPSC15、ChiPSC17、ChiPSC18、ChiPSC19、ChiPSC20、ChiPSC21、ChiPSC23、ChiPSC24、P11012、P11021、P11025及びSA121)。
図2A〜D:
hPSの胚体内胚葉への分化期の2〜3日目のDNA脱メチル化処理を含む基本プロトコールA又はBを用いることで、27個の異なるhPSC株からの未分化幹細胞は、未分化hESC(SA181)及びhiPSC(ChiPSC4)と比較して、幹細胞マーカーOct−4及びNanogの低mRNA発現レベル(図2A、B)及びDEマーカーSox17及びCxcr4の高レベル(図2C、D)を示す、高度に均一なDEへと分化させることができた。
基本プロトコールA(hPSC株P11032及びSA181)又はB(hPSC株P11012)に続いて、3個の異なるhPSC株に由来する細胞は、hPSの胚体内胚葉への分化期、2〜3日目に、10nMの5−アザ−2−デオキシシチジン又は1μMの5−アザシチジンのいずれかで処理された。
図3:
A,B)脱メチル化剤で処理しない場合、3種のhPSC株P11032、SA181及びP11012は、幹細胞マーカーOct4及びNanogの比較的高いmRNA発現をともなう不均一なDEを産生した(図3A,B)。DNA脱メチル化剤5−アザ−2−デオキシシチジン(5azadC)及び5−アザシチジン(5azaC)での処理は、Oct4及びNanog mRNAを有意に減少させ(図3A,B)、これにより、これら3種のhPSC株からの均一なDE集団の誘導を可能にした。
C,D)10nMの5−アザ−2−デオキシシチジン又は1μMの5−アザシチジンでの処理時に、DEマーカーSox17及びCxcr4のmRNA発現での有意な変化を観察することはできなかった(図3C,D)。
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Claims (13)
- 1nMないし1μMの少なくとも1種のヌクレオシド類似体タイプのDNA脱メチル化剤とアクチビンとを含む、哺乳類多能性幹細胞から胚体内胚葉細胞を産生するための組成物。
- 前記DNA脱メチル化剤がシチジン類似体である請求項1に記載の組成物。
- 前記DNA脱メチル化剤が5−アザ−2−デオキシシチジン(デシタビン)、5−アザシチジン(アザシチジン)、ゼブラリン、プソイドイソシチジン、5−フルオロ−2−デオキシシチジン、5,6−ジヒドロ−5−アザシチジン、2’−デオキシ−5,6−ジヒドロ−5−アザシチジン、6−アザシチジン、2’,2’−ジフルオロ−デオキシシチジン(ゲムシタビン)又はシトシン−ベータ−D−アラビノフラノシドからなる群から選択される請求項1又は2に記載の組成物。
- 前記DNA脱メチル化剤が5−アザ−2−デオキシシチジン(デシタビン)、5−アザシチジン(アザシチジン)、5−フルオロ−2−デオキシシチジン、5,6−ジヒドロ−5−アザシチジン、2’−デオキシ−5,6−ジヒドロ−5−アザシチジン、6−アザシチジン及び2’,2’−ジフルオロ−デオキシシチジン(ゲムシタビン)からなる群から選択される請求項1又は2に記載の組成物。
- 前記DNA脱メチル化剤が5−アザ−2−デオキシシチジン(デシタビン)である請求項1又は2に記載の組成物。
- 前記DNA脱メチル化剤が5−アザシチジン(アザシチジン)である請求項1又は2に記載の組成物。
- 前記アクチビンがアクチビンA又はアクチビンBである請求項1〜6のいずれか1項に記載の組成物。
- 前記DNA脱メチル化剤の濃度が1nMないし500nMの範囲である請求項1〜7のいずれか1項に記載の組成物。
- 前記DNA脱メチル化剤の濃度が1nMないし100nMの範囲である請求項1〜8のいずれか1項に記載の組成物。
- 前記DNA脱メチル化剤の濃度が5nMないし50nMの範囲である請求項1〜9のいずれか1項に記載の組成物。
- 前記DNA脱メチル化剤の濃度が5nMないし15nMの範囲である請求項1〜10のいずれか1項に記載の組成物。
- 前記DNA脱メチル化剤の濃度が7.5nMないし12.5nMの範囲である請求項1〜11のいずれか1項に記載の組成物。
- 哺乳類多能性幹細胞から胚体内胚葉細胞をin vitroで産生するための請求項1〜12のいずれか1項に記載の組成物の使用。
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