JP2017533731A - 合成ペプチドを利用した人工多能性幹細胞の製造方法 - Google Patents
合成ペプチドを利用した人工多能性幹細胞の製造方法 Download PDFInfo
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- JP2017533731A JP2017533731A JP2017543696A JP2017543696A JP2017533731A JP 2017533731 A JP2017533731 A JP 2017533731A JP 2017543696 A JP2017543696 A JP 2017543696A JP 2017543696 A JP2017543696 A JP 2017543696A JP 2017533731 A JP2017533731 A JP 2017533731A
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Abstract
Description
[一般式I]GKCSTRGRKX1X2RRKK
前記X1およびX2は、システイン(C:cysteine)またはメチオニン(M:methionine)である。
[一般式I]GKCSTRGRKX1X2RRKK
前記X1およびX2は、システイン(C:cysteine)またはメチオニン(M:methionine)である。
[一般式I]GKCSTRGRKX1X2RRKK
前記X1およびX2は、システイン(C:cysteine)またはメチオニン(M:methionine)である。
[一般式I]GKCSTRGRKX1X2RRKK
前記X1およびX2は、システイン(C:cysteine)またはメチオニン(M:methionine)である。
[一般式I]GKCSTRGRKX1X2RRKK
前記X1およびX2は、システイン(C:cysteine)またはメチオニン(M:methionine)である。
[一般式I]GKCSTRGRKX1X2RRKK
前記X1およびX2は、システイン(C:cysteine)またはメチオニン(M:methionine)である。
[一般式I]GKCSTRGRKX1X2RRKK
前記X1およびX2は、システイン(C:cysteine)またはメチオニン(M:methionine)である。
P1ペプチド:GKCSTRGRKCCRRKK(配列番号1)
P2ペプチド:GKCSTRGRKCMRRKK(配列番号2)
P3ペプチド:GKCSTRGRKMCRRKK(配列番号3)
本発明において、分化された細胞は、体細胞または前駆細胞であることが好ましく、ヒト由来であることが好ましいが、これに限定されない。
本発明において、体細胞培養にはEMEM(Eagle’s Minimum Essential Medium,ATCC 30−2003)を使用することが好ましく、ペプチドを処理した以後にはEssential 8 Medium(Gibco,A15169−01)を使用することが好ましいが、これに限定されない。
本発明において、リプログラミング遺伝子は、Oct3/4、Sox2、c−Myc、Klf4およびLin28からなる群から選択されたいずれか一つ以上であることが好ましいが、これに限定されない。Oct3/4、Sox2、Klf4、c−MycまたはLin28逆分化因子(reprogramming gene)は分化が終わった細胞を再プログラム化させることができる遺伝子を意味して、特にOct4、Sox2、Klf4およびc−Mycは、Yamanaka因子と呼ばれる。
P1ペプチド(GKCSTRGRKCCRRKK:配列番号1)を合成装置を利用してC末端からF−moc固体相化学合成方法で合成した。すなわち、ブロッキンググループ(Blocking group)でFmoc−(9−Fluorenylmethoxycarbonyl)が結合されたRinkレジン(0.075mmol/g、100〜200mesh、1% DVB crosslinking)を使用して合成して、合成器に50mgのRink Amide MBHAレジンを入れた後、DMFでレジンをスウェリング(swelling)させた後、Fmoc−groupの除去のために20%piperidine/DMF溶液を使用した。C末端から配列毎に0.5M amino acid溶液(溶媒:DMF)、1.0M DIPEA(溶媒:DMF&NMP),0.5M HBTU(溶媒:DMF)を各々5、10、15当量ずつ入れて窒素気流下で1〜2時間反応させた。前記脱保護(deprotection)とカップリング(coupling)段階が終る度にDMFとメタノールで二回ずつ洗浄する過程を経た。最後のアミノ酸をカップリング(coupling)させた後にも、脱保護(deprotection)を行ってFmoc−groupを除去した。
P1ペプチド:GKCSTRGRKCCRRKK(配列番号1)
また、P1ペプチド(配列番号1)のC末端5番目のシステインをメチオニンで置き換えたP2ペプチド(配列番号2)及びP1ペプチド(配列番号1)のC末端6番目のシステインをメチオニンで置き換えたP3ペプチド(配列番号3)を合成装置を利用してF−moc個相化学合成方法で合成した。
P2ペプチド:GKCSTRGRKCMRRKK(配列番号2)
P3ペプチド:GKCSTRGRKMCRRKK(配列番号3)
前記実施例1と同様の合成装置を利用してF−moc個相化学合成方法で合成した。
C1ペプチド:HRRCNKNNKKR(配列番号4)
前記実施例1と同様の合成装置を利用してF−moc個相化学合成方法で合成した。
C2ペプチド:GLRSKSKKFRRPDIQYPDA(配列番号5)
2−1:ALP染色を介した人工多能性幹細胞コロニー個数確認
ペプチド別逆分化誘導効率を確認するために、ヒト組換えビトロネクチンタンパク質(Vitronectin Recombinant Human protein,VTN−N)がコーティングされた6−ウェル(well)を利用して1.5×105個/ウェル(well)のヒト由来皮膚線維芽細胞(hDF:human dermal fibroblasts)に電気穿孔(electroporation)手法でリプログラミング遺伝子DNA(Oct3/4、Sox2、c−Myc、Klf4、Lin28)を2μgずつ導入した。前記逆分化因子は公示の方法に従って、各々異なる三つのepisomal Vectorに分けて製作してp53 shRNAを共に発現させて(pCXLE−hOCT3/4−shp53,pCXLE−hSK,pCXLE−hUL)逆分化幹細胞の確立効率を上げた(Okita K et al.,Nat Methods.8(5):409−12,2011)。逆分化因子が導入された細胞は、EMEM(Eagle’s minimal essential medium,ATCC)培地で培養して、細胞confluencyが50%になる時まで24時間間隔で培地を取り替えた。Confluencyが50%になった時、Essential 8培地に替えて逆分化誘導促進ペプチドと対照群ペプチドを各々10μM処理して、24時間毎に培地の取り替えと同時にペプチドを処理した。ペプチドを24時間間隔で10日間処理して、胚性幹細胞のマーカー(marker)として知られるアルカリホスファターゼ(Alkaline Phosphatase,AP)でペプチド特別な逆分化効率を確認するために、AP染色キットはMiliporeのAlkaline Phosphatase Detection Kitを購入して使用した。培地を除去して4%パラホルムアルデヒド(Paraformaldehyde)を細胞に入れて2分間固定してNaphthol/Fast Red Violet染色剤をウェル(well)に入れて20分以上暗い空間で室温保管して反応させた後、DPBS(Dulbecco’s phosphate buffered saline)で染色剤を洗浄して顕微鏡で染色されたコロニーの個数を確認した。
実施例2−1と同様の方法で、ヒト皮膚細胞(human Dermal fibroblasts)に電気穿孔(electroporataion)手法で逆分化因子を細胞内に導入してビトロネクチン(Vitronectin)がコーティングされた6−ウェル(well)に分注した。以後、免疫蛍光(immunofluorescence,IF)手法を利用して胚性幹細胞マーカー(marker)であるOct4の発現程度を比較した。
実施例2−1と同様の方法で、ヒト皮膚細胞(human Dermal fibroblasts)に電気穿孔(electroporataion)手法で逆分化因子を細胞内に導入してビトロネクチン(Vitronectin)がコーティングされた6−ウェル(well)に分注した。細胞が安定化されたペプチド処理10日後に、スクレーパーを利用して細胞を15mlパルコンチューブ(Falcon tube)に集めた後、1500rpmで3分間遠心分離した。緩衝溶液(PBS)で洗浄後、冷たい緩衝溶液(cold PBS)を利用して1.5×105個の細胞を各々のチューブに移した。チューブに10μg/ml濃度のTHY1(cat:sc−59398)、EPCAM(cat:ab20160)抗体を3%BSA/PBS溶液に希釈して入れて4℃で30分間反応させた後、1500rpmで5分間3回遠心分離した。冷たい緩衝溶液(cold PBS)で洗浄後、ラウンドチューブ(round tube)に移してフローサイトメトリー(flow cytometry,FACS)で分析した。サンプルの測定に先立ち、THY1はヒト由来線維芽細胞(human dermal fibroblasts)を使用して、EPCAMはヒト由来乳腺上皮細胞(human mammary epithelial cell,HMEC)を使用してコントロール基準値を固定した。
Claims (17)
- 下記の一般式Iのアミノ酸配列で表示されるペプチドを有効成分として含有する、分化した細胞からの人工多能性幹細胞への逆分化促進用組成物:
[一般式I]GKCSTRGRKX1X2RRKK
(前記X1およびX2は、システイン(C)またはメチオニン(M)である)。 - 前記ペプチドは、配列番号1〜3で構成された群から選択されたいずれか一つであることを特徴とする請求項1に記載の人工多能性幹細胞への逆分化促進用組成物。
- 前記分化した細胞は、体細胞または前駆細胞であることを特徴とする請求項1に記載の人工多能性幹細胞への逆分化促進用組成物。
- 前記分化した細胞は、ヒト由来であることを特徴とする請求項1に記載の人工多能性幹細胞への逆分化促進用組成物。
- 下記段階を含む分化した細胞からの人工多能性幹細胞の製造方法:
(a)分化した細胞にリプログラミング遺伝子を導入する段階;および
(b)リプログラミング遺伝子が導入された細胞に下記一般式Iのアミノ酸配列で表示されるペプチドを処理して培養する段階:
[一般式I]GKCSTRGRKX1X2RRKK
(前記X1およびX2は、システイン(C)またはメチオニン(M)である)。 - 前記ペプチドは、配列番号1〜3で構成された群から選択されたいずれか一つであることを特徴とする請求項5に記載の人工多能性幹細胞の製造方法。
- 前記ペプチドは、0.01〜100μM濃度であることを特徴とする請求項5に記載の人工多能性幹細胞の製造方法。
- 前記分化した細胞は、体細胞または前駆細胞であることを特徴とする請求項5に記載の人工多能性幹細胞の製造方法。
- 前記分化した細胞は、ヒト由来であることを特徴とする請求項5に記載の人工多能性幹細胞の製造方法。
- 無異種感染(xenopathogen free)または無支持細胞(feeder cell free)条件下で行われることを特徴とする請求項5に記載の人工多能性幹細胞の製造方法。
- 前記リプログラミング遺伝子は、Oct3/4、Sox2、c−Myc、Klf4およびLin28からなる群から選択されたいずれか一つ以上であることを特徴とする請求項5に記載の人工多能性幹細胞の製造方法。
- 前記ペプチドは、NF−κB活性を抑制することを特徴とする請求項5に記載の人工多能性幹細胞の製造方法。
- 前記ペプチドは、間葉系細胞−上皮細胞転移(Mesenchymal−Epithelial transition,MET)を促進することを特徴とする請求項5に記載の人工多能性幹細胞の製造方法。
- 前記(b)段階において、ペプチドを細胞培養培地に直接処理するか、又はバイオマテリアルと混合して処理することを特徴とする請求項5に記載の人工多能性幹細胞の製造方法。
- 前記バイオマテリアルとは、合成高分子または天然高分子であることを特徴とする請求項14に記載の人工多能性幹細胞の製造方法。
- 前記合成高分子は、ポロキサマー、ポリエチレングリコール及びポリプロピレングリコールで構成された群から選択されたいずれか一つであることを特徴とする請求項15に記載の人工多能性幹細胞の製造方法。
- 前記天然高分子は、ビトロネクチン、コラーゲン、ゼラチン、アルギン酸、コンドロイチン硫酸、フィブロネクチン及び細胞外マトリックスタンパク質で構成された群から選択されたいずれか一つであることを特徴とする請求項15に記載の人工多能性幹細胞の製造方法。
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