JPWO2017141900A1 - 多能性幹細胞の神経幹細胞への分化用培地及びその使用 - Google Patents
多能性幹細胞の神経幹細胞への分化用培地及びその使用 Download PDFInfo
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Abstract
Description
[1]多能性幹細胞の由来に関わらず、前記多能性幹細胞の株間又はクローン間のばらつきを解消し、胚様体を形成させずに多能性幹細胞を均一に神経幹細胞に分化させる方法。
[2]前記多能性幹細胞をFibroblast Growth Factor 2(FGF2)、Rho−associated protein kinase(ROCK)阻害剤及びLeukemia Inhibitory Factor(LIF)を有効成分として含有する培地中で培養する工程を備える、[1]に記載の方法。
[3]前記培養する工程を低酸素条件下で行う、[2]に記載の方法。
[4]前記培養する工程の前に、前記多能性幹細胞を細胞1個ずつに解離させる工程を更に備える、[2]又は[3]に記載の方法。
[5]前記多能性幹細胞が血球由来の人工多能性幹細胞である、[1]〜[4]のいずれかに記載の方法。
[6]前記人工多能性幹細胞がT細胞由来である、[5]に記載の方法。
[7][1]〜[6]のいずれかに記載の方法に使用するための、FGF2、ROCK阻害剤及びLIFを有効成分として含有する培地。
[8][1]〜[6]のいずれかに記載の方法で作製された神経幹細胞。
[9]前脳マーカー、及び前脳/中脳マーカーを発現している、[8]に記載の神経幹細胞。
[10]Homeobox B4(HOXB4)を実質的に発現していない、[8]又は[9]に記載の神経幹細胞。
[11]T細胞受容体遺伝子が再編成している、[8]〜[10]のいずれかに記載の神経幹細胞。
[12]多能性幹細胞を神経幹細胞に分化させる効率を向上させる方法であって、多能性幹細胞をFGF2、ROCK阻害剤及びLIFを有効成分として含有する培地中で培養する工程を備える、方法。
[13]前記多能性幹細胞を神経幹細胞に分化させる、向上された効率が、皮膚繊維芽細胞由来の人工多能性幹細胞を神経幹細胞に分化させる効率と同等の効率である、[12]に記載の方法。
[14]前記培養する工程を低酸素条件下で行う、[12]又は[13]に記載の方法。
[15]前記培養する工程の前に、前記多能性幹細胞を細胞1個ずつに解離させる工程を更に備える、[12]〜[14]のいずれかに記載の方法。
[16]前記多能性幹細胞が血球由来の人工多能性幹細胞である、[12]〜[15]のいずれかに記載の方法。
[17]前記人工多能性幹細胞がT細胞由来である、[16]に記載の方法。
[18][12]〜[17]のいずれかに記載の方法に使用するための、FGF2、ROCK阻害剤及びLIFを有効成分として含有する培地。
[A2]前記多能性幹細胞が人工多能性幹細胞である、[A1]に記載の分化用培地。
[A3]多能性幹細胞を[A1]又は[A2]に記載の培地中で培養する工程を備える、神経幹細胞の製造方法。
[A4]前記多能性幹細胞が人工多能性幹細胞である、[A3]に記載の製造方法。
[A5]前記培養する工程を低酸素環境下で行う、[A3]又は[A4]に記載の製造方法。
[A6]前記培養する工程の前に、前記多能性幹細胞を細胞1個ずつに解離させる工程を更に備える、[A3]〜[A5]のいずれかに記載の製造方法。
[A7]HOXB4を実質的に発現していない神経幹細胞。
[A8]T細胞受容体遺伝子が再編成している、[A7]に記載の神経幹細胞。
1実施形態において、本発明は、多能性幹細胞の由来に関わらず、前記多能性幹細胞の株間又はクローン間のばらつきを解消し、胚様体を形成させずに多能性幹細胞を均一に神経幹細胞に分化させる方法を提供する。
1実施形態において、本発明は、多能性幹細胞を神経幹細胞に分化させる効率を向上させる方法であって、多能性幹細胞をFGF2、ROCK阻害剤及びLIFを有効成分として含有する培地中で培養する工程を備える、方法を提供する。
1実施形態において、本発明は、FGF2、ROCK阻害剤及びLIFを有効成分として含有する、多能性幹細胞の神経幹細胞への分化用培地を提供する。
1実施形態において、本発明は、多能性幹細胞を上述した培地中で培養する工程を備える、神経幹細胞の製造方法を提供する。
1実施形態において、本発明は、HOXB4を実質的に発現していない神経幹細胞を提供する。本実施形態の神経幹細胞は、上述した神経幹細胞の製造方法により製造することができる。
(T細胞及び皮膚繊維芽細胞からのiPSCの作製)
健常人から採取したT細胞及び皮膚繊維芽細胞からiPSCを作製した。まず、健常人由来のCD3陽性リンパ球に、OCT4、SOX2、KLF4、L−MYC、LIN28、EBNA1、及びp53に対するshRNA又はドミナントネガティブp53を含むエピソームベクターを導入し、ヒトT細胞由来iPSC(T−cell−derived iPSC、以下、「TiPSC」という場合がある。)株であるeTKA4及びeTKA5を樹立した。
[実験例2]
(従来法によるiPSCの神経幹細胞への分化)
発明者らは、複数種類のaHDF−iPSC株及び複数種類のTiPSC株を、従来法であるEB法及びDSi−EB法により神経幹細胞に分化させた。aHDF−iPSC株としては、上述したKA11、KA23、eKA3及びeKA4を使用した。また、TiPSCとしては、上述したeTKA4、eTKA5、TKA7、TKA14、TKA4及びTKA9を使用した。
EB法においては、まず、細胞1個ずつに解離させた各iPSCを培養してEBを形成させた。続いて、EBを、神経幹細胞/神経前駆細胞に分化させた。
続いて、SMADシグナル伝達経路の阻害剤である、SB431542及びNogginの存在下でEBを形成させるDSi−EB法による検討を行った。SB431542及びNogginは、デュアルSMAD阻害(dual SMAD inhibition、DSi)を誘導することが知られている(例えば、Chambers S. M., et al., Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMADsignaling, Nat. Biotechnol., 27 (3), 275-280, 2009.を参照。)
[実験例3]
発明者らは、iPSCの神経幹細胞への分化において、EBの形成を含まない新たな方法(direct−neurosphere法、以下、「dNS法」という場合がある。
)を開発した。図4は、dNS法の手順を示す図である。dNS法では、細胞1個ずつに解離させたiPSCを、低酸素条件下において、FGF2、Y27632及びLIFを含有する無血清培地(以下、「NS培地」という場合がある。)で培養する。後述するように、dNS法によれば約14日でiPSCから神経幹細胞塊を形成することができる。具体的なNS培地の組成を下記表1に示す。
[実験例4]
dNS法において、NS培地の存在下又は非存在下、及び低酸素条件の存在下又は非存在下の条件で、複数種類のaHDF−iPSC株及び複数種類のTiPSC株を神経幹細胞に分化させ、dNS法におけるNS培地及び低酸素条件の影響を検討した。
[実験例5]
続いて、発明者らは、TiPSC由来の神経幹細胞塊を、aHDF−iPSC由来の神経幹細胞塊と同程度に機能的なニューロン及びニューロンサブタイプに分化することができるか否かについて検討した。
Claims (18)
- 多能性幹細胞の由来に関わらず、前記多能性幹細胞の株間又はクローン間のばらつきを解消し、胚様体を形成させずに多能性幹細胞を均一に神経幹細胞に分化させる方法。
- 前記多能性幹細胞をFibroblast Growth Factor 2(FGF2)、Rho−associated protein kinase(ROCK)阻害剤及びLeukemia Inhibitory Factor(LIF)を有効成分として含有する培地中で培養する工程を備える、請求項1に記載の方法。
- 前記培養する工程を低酸素条件下で行う、請求項2に記載の方法。
- 前記培養する工程の前に、前記多能性幹細胞を細胞1個ずつに解離させる工程を更に備える、請求項2又は3に記載の方法。
- 前記多能性幹細胞が血球由来の人工多能性幹細胞である、請求項1〜4のいずれか一項に記載の方法。
- 前記人工多能性幹細胞がT細胞由来である、請求項5に記載の方法。
- 請求項1〜6のいずれか一項に記載の方法に使用するための、FGF2、ROCK阻害剤及びLIFを有効成分として含有する培地。
- 請求項1〜6のいずれか一項に記載の方法で作製された神経幹細胞。
- 前脳マーカー、及び前脳/中脳マーカーを発現している、請求項8に記載の神経幹細胞。
- Homeobox B4(HOXB4)を実質的に発現していない、請求項8又は9に記載の神経幹細胞。
- T細胞受容体遺伝子が再編成している、請求項8〜10のいずれか一項に記載の神経幹細胞。
- 多能性幹細胞を神経幹細胞に分化させる効率を向上させる方法であって、多能性幹細胞をFGF2、ROCK阻害剤及びLIFを有効成分として含有する培地中で培養する工程を備える、方法。
- 前記多能性幹細胞を神経幹細胞に分化させる、向上された効率が、皮膚繊維芽細胞由来の人工多能性幹細胞を神経幹細胞に分化させる効率と同等の効率である、請求項12に記載の方法。
- 前記培養する工程を低酸素条件下で行う、請求項12又は13に記載の方法。
- 前記培養する工程の前に、前記多能性幹細胞を細胞1個ずつに解離させる工程を更に備える、請求項12〜14のいずれか一項に記載の方法。
- 前記多能性幹細胞が血球由来の人工多能性幹細胞である、請求項12〜15のいずれか一項に記載の方法。
- 前記人工多能性幹細胞がT細胞由来である、請求項16に記載の方法。
- 請求項12〜17のいずれか一項に記載の方法に使用するための、FGF2、ROCK阻害剤及びLIFを有効成分として含有する培地。
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WO2016159179A1 (ja) * | 2015-03-30 | 2016-10-06 | 味の素株式会社 | ヒト血清アルブミンを含む神経幹細胞増殖培地 |
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WO2016159179A1 (ja) * | 2015-03-30 | 2016-10-06 | 味の素株式会社 | ヒト血清アルブミンを含む神経幹細胞増殖培地 |
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