JP5886212B2 - iPS細胞とその製造法 - Google Patents
iPS細胞とその製造法 Download PDFInfo
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Description
1.バクテリアDNAなどの外来遺伝子が宿主細胞のゲノムに組み込まれる恐れがある。
2.従来の方法によりできたiPS細胞は、一定の頻度で癌化し、安全面での問題がある。
3.外来遺伝子の過剰発現でできたiPS細胞では、多能性以外の影響が予想できない。
項1. 少なくとも1種のコネキシン抑制因子と少なくとも1種のTGFβシグナリング抑制因子を細胞に作用させることを特徴とするiPS細胞を作製する方法。
項2. コネキシン抑制因子がコネキシン43抑制因子である項1に記載の方法。
項3. 前記コネキシン抑制因子が、コネキシンに対するsiRNA、shRNA、miRNA、アンチセンスRNA、ペプチド、抗体もしくはリボザイム、又は細胞内でこれらのRNA分子を放出できる発現ベクターであることを特徴とする、項1または2に記載の方法。
項4. 前記TGFβシグナリング抑制因子がTGF-βレセプターI阻害剤である項1〜3のいずれかに記載の方法。
項5. 前記TGF-βレセプター阻害剤がSB-431542、A-83-01またはALK5 Inhibitorである項1〜4のいずれかに記載の方法。
項6. 少なくとも1種のコネキシン抑制因子を含むiPS細胞。
項7. コネキシン抑制因子がコネキシン43抑制因子である項6に記載のiPS細胞。
項8. 前記コネキシン抑制因子が、コネキシンに対するsiRNA、shRNA、miRNA、アンチセンスRNA、ペプチド、抗体もしくはリボザイム、又は細胞内でこれらのRNA分子を放出できる発現ベクターであることを特徴とする、項6または7に記載のiPS細胞。
項9. コネキシン抑制因子及びTGFβシグナリング抑制因子からなる群から選ばれる少なくとも1種の抑制因子を含むiPS細胞誘導剤。
項10. コネキシン抑制因子及びTGFβシグナリング抑制因子からなる群から選ばれる少なくとも1種の抑制因子を含むiPS細胞誘導用培地。
項11. コネキシン抑制因子及びTGFβシグナリング抑制因子からなる群から選ばれる少なくとも1種の抑制因子を含むiPS細胞誘導用キット。
1. 多能性細胞マーカー因子の発現が遺伝子レベル、タンパク質レベルで保たれている。
2. マウス皮下へ移植することにより、テラトーマが形成され、腺管、軟骨、神経など三胚葉全ての細胞が分化誘導できる。
3. 胚葉体を形成し、分化誘導条件により、内皮細胞、神経細胞、心筋細胞へとそれぞれ分化可能で、分化誘導できた心筋細胞が生理的に機能する。
4. キメラマウスの作製が可能である.
センス: 5’-CAA UUC UUC UUG CCG CAA TT-3’ (配列番号1)
アンチセンス: 5’-UUG CGG CAA GAA GAA UUG TT-3’ (配列番号2)
抗コネキシンポリヌクレオチドには、コネキシンアンチセンスポリヌクレオチドおよびコネキシン発現を下方制御させることができる機能を有するポリヌクレオチドが含まれる。他の適切な抗コネキシンポリヌクレオチドには、RNAiポリヌクレオチドおよびsiRNAポリヌクレオチド、shRNAポリヌクレオチドが含まれる。
GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC (配列番号3);
GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC (配列番号4);
GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT (配列番号5).
5' TCC TGA GCA ATA CCT AAC GAA CAA ATA (connexin 26) (配列番号6);
5' CGT CCG AGC CCA GAA AGA TGA GGT C (connexin 31.1) (配列番号11);
5' TTT CTT TTC TAT GTG CTG TTG GTG A (connexin 32) (配列番号14).
5' CAT CTC CTT GGT GCT CAA CC 3' (connexin 37) (配列番号 7);
5' CTG AAG TCG ACT TGG CTT GG 3' (connexin 37) (配列番号 8);
5' CTC AGA TAG TGG CCA GAA TGC 3' (connexin 30) (配列番号 9);
5' TTG TCC AGG TGA CTC CAA GG 3' (connexin 30) (配列番号 10);
5' AGA GGC GCA CGT GAG ACA C 3' (connexin 31.1) (配列番号 12);
5' TGA AGA CAA TGA AGA TGT T 3' (connexin 31.1) (配列番号 13).
例えば、ポリヌクレオチドは、コネキシンmRNA中の配列の相補物のホモログであり得る。かかるポリヌクレオチドは、典型的には、(相同配列の)例えば少なくとも約15、少なくとも約20、少なくとも約40、少なくとも約100を超える連続ヌクレオチドの領域にわたって、関連配列と少なくとも約90%、少なくとも約95%、少なくとも約97%、少なくとも約98%、または少なくとも約99%相同である。
結合タンパク質(ペプチド、ペプチド模倣物、抗体、および抗体フラグメントなどが含まれる)はまた、適切な抑制因子である。
FEVAFLLIQWI (配列番号15)
LLIQWYIGFSL (配列番号16)
SLSAVYTCKRDPCPHQ (配列番号17)
VDCFLSRPTEKT (配列番号18)
SRPTEKTIFII (配列番号19)
LGTAVESAWGDEQ (配列番号20)
QSAFRCNTQQPG (配列番号21)
QQPGCENVCYDK (配列番号22)
VCYDKSFPISHVR (配列番号23)
Cx32にターゲティングされるペプチド(配列番号34は、Cx32/Cx43)
ESVWGDEKSSFI (配列番号 24)
ICNTLQPGCNSV (配列番号 25)
SVCYDHFFPISH (配列番号 26)
RLVKCEAFPCPNTVDCFVSRPTEKT (配列番号 27)
VKCEAFPCPNTV (配列番号 28)
VDCFVSRPTEKT (配列番号 29)
VCYDHFFPISHVR (配列番号 30)
VWGDEKSSFICNTLQPGY (配列番号 31)
DEKSSFICNTLQPGY (配列番号 32)
SRPTEKTVFTV (配列番号 33)
SRPTEKT (配列番号 34)
ICNTLQPGCNSV (配列番号 35)
Cx40にターゲティングされるペプチド
FLDTLHVCRRSPCPHP (配列番号 36)
Cx43にターゲティングされるペプチド
KRDPCHQVDCFLSRPTEK (配列番号 37)
huCx26 KEVWGDEQADFVCNTLQPGCKNVCYDHYFPISHIR (配列番号38)
huCx30 QEVWGDEQEDFVCNTLQPGCKNVCYDHFFPVSHIR (配列番号 39)
huCx30.3 EEVWDDEQKDFVCNTKQPGCPNVCYDEFFPVSHVR (配列番号 40)
huCx31 ERVWGDEQKDFDCNTKQPGCTNVCYDNYFPISNIR (配列番号 41)
huCx31.1 ERVWSDDHKDFDCNTRQPGCSNVCFDEFFPVSHVR (配列番号 42)
huCx32 ESVWGDEKSSFICNTLQPGCNSVCYDQFFPISHVR (配列番号 43)
huCx36 ESVWGDEQSDFECNTAQPGCTNVCYDQAFPISHIR (配列番号 44)
huCx37 ESVWGDEQSDFECNTAQPGCTNVCYDQAFPISHIR (配列番号 45)
huCx40.1 RPVYQDEQERFVCNTLQPGCANVCYDVFS PVSHLR (配列番号 46)
huCx43 ESAWGDEQSAFRCNTQQPGCENVCYDKSFPISHVR (配列番号 47)
huCx46 EDVWGDEQSDFTCNTQQPGCBNVCYBRAFPISHIR (配列番号 48)
huCx46.6 EAIYSDEQAKFTCNTRQPGCDNVCYDAFAPLSHVR (配列番号 49)
huCx40 ESSWGDEQADFRCDTIQPGCQNVCTDQAFPISHIR (配列番号 50)
huCx45 GESIYYDEQSKFVCNTEQPGCENVCYDAFAPLSHVR (配列番号 51)
huCx26 MYVFYVMYDGFSMQRLVKCNAWPCPNTVDCFVSRPTEKT (配列番号 52)
huCx30 MYVFYFLYNGYHLPWVLKCGIDPCPNLVDCFISRPTEKT (配列番号 53)
huCx30.3 LYIFHRLYKDYDMPRVVACSVEPCPHTVDCYISRPTEKK (配列番号 54)
huCx31 LYLLHTLWHGFNMPRLVQCANVAPCPNIVDCYIARPTEKK (配列番号 55)
huCx31.1 LYVFHSFYPKYILPPVVKCHADPCPNIVDCFISKPSEKN (配列番号 56)
huCx32 MYVFYLLYPGYAMVRLVKCDVYPCPNTVDCFVSRPTEKT 配列番号 57)
huCx36 LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKT (配列番号 58)
huCx37 LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKT (配列番号 59)
huCx40.1 GALHYFLFGFLAPKKFPCTRPPCTGVVDCYVSRPTSKS (配列番号 60)
huCx43 LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKT (配列番号 61)
huCx46 IAGQYFL YGFELKPLYRCDRWPCPNTVDCFISRPTEKT (配列番号 62)
huCx46.6 LVGQYLLYGFEVRPFFPCSRQPCPHVVDCFVSRPTEKT (配列番号 63)
huCx40 IVGQYFIYGIFLTTLHVCRRSPCPHPVNCYVSRPTEKN (配列番号 64)
huCx45 LIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKT (配列番号 65)
huCx43 LLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTIFII(配列番号66)
huCx26 MYVFYVMYDGFSMQRLVKCNAWPCPNTVDCFVSRPTEKTVFTV(配列番号 67)
huCx30 YVFYFLYNGYHLPWVLKCGIDPCPNLVDCFISRPTEKTVFTI(配列番号 68)
huCx30.3 LYIFHRLYKDYDMPRVVACSVEPCPHTVDCYISRPTEKKVFTY(配列番号 69)
huCx31 LYLLHTLWHGFNMPRLVQCANVAPCPNIVDCYIARPTEKKTY(配列番号70)
huCx31.1 LYVFHSFYPKYILPPVVKCHADPCPNrVDCFISKPSEKNIFTL(配列番号 71)
huCx32 MYVFYLLYPGYAMVRLVKCDVYPCPNTVDCFVSRPTEKTVFTV(配列番号 72)
huCx36 LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKTIFII(配列番号 73)
huCx37 LYGWTMEPVFVCQRAPCPYLVDCFVSRPTEKTIFII(配列番号 74)
huCx40.1 GALHYFLFGFLAPKKFPCTRPPCTGVVDCYVSRPTEKSLLML(配列番号 75)
huCx46 IAGQYFLYGFELKPLYRCDRWPCPNTVDCFISRPTEKTIFII(配列番号 76)
huCx46.6 LVGQYLLYGFEVRPFFPCSRQPCPHVVDCFVSRPTEKTVFLL(配列番号 77)
huCx40 IVGQYFIYGIFLTTLHVCRRSPCPHPVNCYSRPTEKNVFIV(配列番号 78)
huCx45 LIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKTIFLLC配列番号 79)
LGTAAESSWGDEQADFRCDTIQPGCQNVCTDQAFPISHIRFWVLQ (配列番号80)
LGTAAESSWGDEQA (配列番号81)
DEQADFRCDTIQP (配列番号82)
TIQPGCQNVCTDQ (配列番号83)
VCTDQAFPISHIR (配列番号84)
AFPISHIRFWVLQ (配列番号85)
MEVGFIVGQYFIYGIFLTTLHVCRRSPCPHPVNCYVSRPTEKNVFIV (配列番号86 )
MEVGFIVGQYF (配列番号87 )
IVGQYFIYGIFL (配列番号88)
GIFLTTLHVCRRSP (配列番号89)
RRSPCPHPVNCY (配列番号90)
VNCYVSRPTEKN (配列番号91)
SRPTEKNVFIV (配列番号92)
LTAVGGESIYYDEQSKFVCNTEQPGCENVCYDAFAPLSHVRFWVFQ (配列番号93)
LTAVGGESIYYDEQS (配列番号 94)
DEQSKFVCNTEQP (配列番号 95)
TEQPGCENVCYDA (配列番号 96)
VCYDAFAPLSHVR (配列番号 97)
APLSHVRFWVFQ (配列番号 98)
FEVGFLIGQYFLYGFQVHPFYVCSRLPCHPKIDCFISRPTEKTIFLL (配列番号 99)
FEVGFLIGQYF (配列番号 100)
LIGQYFLYGFQV (配列番号 101)
GFQVHPFYVCSRLP (配列番号 102)
SRLPCHPKIDCF (配列番号 103)
IDCFISRPTEKT (配列番号 104)
SRPTEKTIFLL (配列番号 105)
YVCSRLPCHP (配列番号 106),
QVHPFYVCSRL (配列番号 107),
FEVGFLIGQYFLY (配列番号 108),
GQYFLYGFQVHP (配列番号109),
GFQVHPFYVCSR (配列番号 110),
AVGGESIYYDEQ (配列番号111),
YDEQSKFVCNTE (配列番号 112),
NTEQPGCENVCY (配列番号 113),
CYDAFAPLSHVR (配列番号 114),
FAPLSHVRFWVF (配列番号 115)
LIGQY (配列番号 116),
QVHPF (配列番号 117),
YVCSR (配列番号 118),
SRLPC (配列番号 119),
LPCHP (配列番号 120)
GESIY (配列番号 121),
YDEQSK (配列番号122),
SKFVCN (配列番号 123),
TEQPGCEN (配列番号 124),
VCYDAFAP (配列番号125),
LSHVRFWVFQ (配列番号126)
(1)Receptor I(ALK 5) inhibitor:
SB425342; A-83-01; ALK5 inhibitor.
(2)Signal transduction inhibitor:
Inhibitor Smad: Smad7がTβR Iに結合し、Smad2及びSmad3のTβR Iへの結合を妨害する。
実施例1:新生仔マウスの心室に由来する心筋細胞の初期化
対象と方法
1)心筋細胞の単離
生後二日後マウス新生仔(C57/BL6及びC57BL/6-Tg (CAG-EGFP))から従来法により心室部分を残した心臓を約2mm×2mmの大きさに解剖用ハサミで細切した。それらを0.2% コラゲナーゼ(Collagenase Type II, Worthington, アメリカ, PBSに溶解)にて、心筋細胞を解離した。解離した心筋細胞を遠心分離し、10% FBS を添加したDMEM培養液で、37℃、5% CO2の条件で45分間インキュベートした後、上清を再度、同条件でインキュベートし、これを4回行い、線維芽細胞を除去し、心筋細胞を分離した。
6 cm dishに2×105心筋細胞を撒き、上述したDMEM培地で一週間培養した後、10 μM TGF-βレセプター阻害剤であるSB-431542 (Tocris, アメリカ) か100nM siRNA Cx43またはその両方を含んだ15% FBS、マウスES培地でそれぞれ心筋細胞の初期化選択を行った。
使用したマウスCx43 siRNA配列は、次の通りである。
antisense: 5’-UUG CGG CAG GAG GAA UUG TT-3’
(i).ES細胞初期マーカーの細胞免疫染色
10 μM SB-431542 と100nM siRNA Cx43両方添加したES培地条件で選択培養した心筋細胞から約2週間で2個のiPS細胞コロニーが得られた。フィーダー細胞上でマウスES細胞培養条件と同様の条件で培養して増殖したiPS様細胞を免疫染色など及びTeratoma形成確認に用いた。
(ii).Teratoma作製
2×105のiPS様細胞をSCIDマウスの皮下に移植し、5週間後、腫瘍を摘出し、HE染色により、奇形腫を形成するかを組織学的に確認した。
(iii).キメラマウスの作成
iPS様細胞をICRマウス由来の8細胞期の受精卵に移植した後、ICRマウスに戻し、キメラマウスができるかを確認した。
1.ES初期マーカー遺伝子の発現
本発明で作製したマウス心筋細胞由来の2個のiPS細胞、siPSCs (silencing-induced pluripotent stem cells) が形態的にマウスES細胞コロニーに類似し(図 1a)、ALP活性が確認でき(図 1b)、多能性マーカー因子、Nanog, Oct4, Sox2, SSEA1の発現がタンパク質レベルで確認できた(図 1c)。また、各種多能性マーカー遺伝子の発現が遺伝子レベルでも確認できた(図 1d)。更にEGFPマウス心筋細胞からのiPS様細胞の誘導も確認できた(図 2)。
iPSCsをSCIDマウスの皮下に移植すると5週間後、外胚葉である上皮と神経、中胚葉である筋肉と軟骨、内胚葉である腺管などの三胚葉系の様々な組織からなるTeratomaを形成した(図 1e)。なお、データは示さないが、siPSCsが胚葉体を形成し、分化誘導条件により神経細胞、心筋細胞、内皮細胞へとそれぞれ分化可能なことが確認できた。心筋細胞では拍動と伝導も認められた。また、免疫抗体法、RT-PCRにより、分化誘導を行った細胞では三胚葉マーカー遺伝子に発現がタンパク質レベルと遺伝子レベルで共に確認できた。
本発明で作製したマウス心筋細胞由来の2個のiPS細胞を胚盤胞への注入により、iPS細胞由来の細胞が混在した成体キメラマウスを得ることができたことから、ES細胞と同様の分化万能性を持つと言える(図3)。
Claims (11)
- コネキシン43、コネキシン40、及びコネキシン45からなる群より選択されるコネキシンの少なくとも1種に対するコネキシン抑制因子と少なくとも1種のTGFβシグナリング抑制因子を心筋細胞に作用させる;又は
コネキシン43、コネキシン26、コネキシン32、及びコネキシン45からなる群より選択されるコネキシンの少なくとも1種に対するコネキシン抑制因子と少なくとも1種のTGFβシグナリング抑制因子を新生仔又は新生児から得た細胞に作用させる;
ことを特徴とするiPS細胞を作製する方法。 - コネキシン抑制因子がコネキシン43抑制因子である請求項1に記載の方法。
- 前記コネキシン抑制因子が、コネキシンに対するsiRNA、shRNA、miRNA、アンチセンスRNA、ペプチド、抗体もしくはリボザイム、又は細胞内でこれらのRNA分子を放出できる発現ベクターであることを特徴とする、請求項1に記載の方法。
- 前記TGFβシグナリング抑制因子がTGF-βレセプターI阻害剤である請求項1に記載の方法。
- 前記TGF-βレセプター阻害剤がSB-431542、A-83-01またはALK5 Inhibitorである請求項1に記載の方法。
- コネキシン43、コネキシン26、コネキシン32、コネキシン40、及びコネキシン45からなる群より選択されるコネキシンの少なくとも1種に対するコネキシン抑制因子を含むiPS細胞。
- コネキシン抑制因子がコネキシン43抑制因子である請求項6に記載のiPS細胞。
- 前記コネキシン抑制因子が、コネキシンに対するsiRNA、shRNA、miRNA、アンチセンスRNA、ペプチド、抗体もしくはリボザイム、又は細胞内でこれらのRNA分子を放出できる発現ベクターであることを特徴とする、請求項6に記載のiPS細胞。
- コネキシン43、コネキシン26、コネキシン32、コネキシン40、及びコネキシン45からなる群より選択されるコネキシンの少なくとも1種に対するコネキシン抑制因子並びにTGFβシグナリング抑制因子を含むiPS細胞誘導剤。
- コネキシン43、コネキシン26、コネキシン32、コネキシン40、及びコネキシン45からなる群より選択されるコネキシンの少なくとも1種に対するコネキシン抑制因子並びにTGFβシグナリング抑制因子を含むiPS細胞誘導用培地。
- コネキシン43、コネキシン26、コネキシン32、コネキシン40、及びコネキシン45からなる群より選択されるコネキシンの少なくとも1種に対するコネキシン抑制因子並びにTGFβシグナリング抑制因子を含むiPS細胞誘導用キット。
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- 2011-12-27 KR KR1020137019315A patent/KR20130131414A/ko not_active Application Discontinuation
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- 2011-12-27 EP EP11853648.1A patent/EP2660314A4/en not_active Withdrawn
- 2011-12-27 JP JP2012550970A patent/JP5886212B2/ja active Active
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KR20130131414A (ko) | 2013-12-03 |
EP2660314A9 (en) | 2015-04-01 |
CA2823154A1 (en) | 2012-07-05 |
EP2660314A1 (en) | 2013-11-06 |
US20120214234A1 (en) | 2012-08-23 |
US8956868B2 (en) | 2015-02-17 |
CN103429732B (zh) | 2015-09-16 |
WO2012090997A1 (ja) | 2012-07-05 |
EP2660314A4 (en) | 2014-09-03 |
JPWO2012090997A1 (ja) | 2014-06-05 |
CN103429732A (zh) | 2013-12-04 |
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