JP6766032B2 - ニューロン細胞及び筋細胞を産生するための方法 - Google Patents
ニューロン細胞及び筋細胞を産生するための方法 Download PDFInfo
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Description
本発明は、人工多能性幹細胞を含めた多能性幹細胞からニューロン細胞及び筋細胞を産生するための方法に関する。
多能性幹細胞は、身体のほぼあらゆる細胞に分化する潜在性を有し、自己再生することができる。最近になって、人工多能性幹細胞(iPSC)の利用可能性は、培養状態でのその無限の増殖能と、任意の異なるヒト細胞型へのその分化能とともに、我々の疾患の病因の理解を改善するために、そして細胞療法又は組織生産を含めた新しい治療法を計画及び試験するためにも、潜在的に貴重な材料源を提供した。実際にごく最近まで、ヒト疾患に特異的な多能性細胞は、5〜6日後の胚の内部細胞塊から得られた既存のヒト胚性幹細胞(hESC)の遺伝的改変か、着床前遺伝子診断により検出可能な単一遺伝子疾患を有する胚からの新しいhESCの産生かによってのみ作製することができた。これらの方法は、非常に限定的であり、わずかな疾患だけがこの方法で研究されている。したがって、最近の人工多能性幹細胞(iPSC)の開発は、いくつかのヒトの疾患の理解及びモデル化に新たな将来性をもたらした。
本発明者らは、本明細書において、多能性細胞からニューロン細胞及び筋管への分化のための新規で効率的な方法を記載する。有利なことには、該方法は、たとえフィーダー細胞層上に成長した多能性細胞にも代替的に適用できるとしても、フィーダー細胞層を必要としない。そのうえ、他の公表されたプロトコールに比べて、本発明の方法は、ニューロン又は筋細胞生成のための胚様体形成も、ニューロン生成のためのニューロンのロゼット(原始神経上皮細胞)又はニューロスフェア形成も必要としないが、これらは細胞集団の純度を改変し得る(Hitoshi et al., Genes Dev. 2004;18:1806-1811; Liu et al., Nat Protoc. 2013;8:1670-1679; Lie et al., Methods Mol Biol. 2012;873:237-246)。本発明の方法は、細胞恒常性を撹乱し得る薬物添加(Li et al., Proc Natl Acad Sci U S A. 2011;108:8299-8304; Menendez et al., Proc Natl Acad Sci U S A. 2011;108:19240-19245; Yan et al., Stem Cells Transl Med. 2013;2:862-870; Chambers et al., Nat Biotechnol. 2009;27:275-280; Surmacz et al., Stem Cells. 2012;30:1875-1884)も、利用可能な分化済み細胞の量を制限する細胞選別又は異所性導入遺伝子発現(Darabi et al., Cell stem cell. 2012;10(5):610-9; Tedesco et al., Sci Transl Med. 2012;4(140):140ra89; Barberi et al., Nature medicine. 2007;13(5):642-8; Awaya et al., PLoS One. 2012;7(12):e51638; Darabi et al., Stem Cells. 2011;29(5):777-90)も必要としない。そのうえ、本発明の方法は、最初の分化段階でサイトカインを2種だけ使用する必要があり、大量のニューロン前駆細胞及び筋前駆細胞を10〜15日で生じ、その細胞を維持し、定期的に増殖させ、又はさらに分化させることができる。以前に公表されたプロトコールによると、FGFが、細胞を未分化状態で維持するために使用された(US 2005/153445)、又は神経前駆細胞を成長させるために最初に使用されたが、次に、細胞分化を誘導するためにとりわけこの因子を抑制するように提唱されている(US 2002/090723)。しかし、以前のプロトコールは、EGFが多能性細胞をニューロン細胞系列に誘導及び成長させるための重要な因子であり得、EGFをFGFと一緒に培地から抑制することがこれらの細胞をニューロン又は筋管にさらに分化させることに有利であると示唆していない。そのうえ、当業者ならば、細胞をEGF中で培養し、次にこの因子を培地から取り除くことよりもむしろ、Yanら、2013(前記)によって提供されるようにBDNF及びGDNFを使用することによってそのようなプロトコールが改善されると予期するであろう。
a)多能性幹細胞からニューロン細胞系列又は筋細胞系列に誘導された細胞であって、フィブロネクチンでコーティングされた支持体上の、FGFファミリータンパク質のメンバー及びEGFを含む培地中で維持された細胞を提供する段階;並びに
b)ニューロンを得るためにラミニンでコーティングされた支持体上、又は筋管若しくは筋管とニューロンとの混合物を得るためにフィブロネクチンでコーティングされた支持体上のいずれかで、FGFファミリータンパク質のいかなるメンバーもEGFも欠如する培地中で、誘導された細胞を培養する段階
を含む、ニューロン又は筋管を産生するための方法を提供する。
i. bFGF、EGF及びDMSOを含有する培地中で多能性幹細胞を培養する段階;次に
ii. bFGF及びEGFを含有し、DMSOを欠如する培地中で細胞を培養する段階;並びに次に
iii. フィブロネクチンでコーティングされた支持体上で、bFGF及びEGFを含有し、DMSOを欠如する培地中で細胞を培養する段階
によって入手可能である。
とりわけ、本発明に関する一般的な方法について、例えば「Molecular Cloning: A Laboratory Manual, 2nd Ed.」(Sambrook et al., 1989)、Animal Cell Culture(R. I. Freshney, ed., 1987))、Methods in Enzymologyのシリーズ(Academic Press)、Gene Transfer Vectors for Mammalian Cells(J. M. Miller & M. P. Calos, eds., 1987);「Current Protocols in Molecular Biology and Short Protocols in Molecular Biology, 3rd Ed.」(F. M. Ausubel et al., eds., 1987 & 1995);Recombinant DNA Methodology II(R. Wu ed., Academic Press 1995)を含めた周知の教科書が参照される。
実験手順
ヒト線維芽細胞のhiPSCへの再プログラミング
初代皮膚線維芽細胞に山中4因子OCT4、KLF4、SOX2及びc−MYC(OKSM)をコードするレンチウイルス(STEMCCA−OKSM多シストロン性ベクター、Millipore)を感染多重度(MOI)5〜20で感染させた後、ヒトiPSCが産生された。
ニューロン前駆細胞の誘導:35mmマトリゲルコーティングプレート(プレート1枚あたりhiPSC約2×106個)上のmTeSR(商標)1培地中で成長した成熟hiPSCコロニーに分化を行った。1日目に、mTeSR(商標)1培地を、2% DMSO(Sigma-Aldrich, cat, No. D2438)を添加した分化培地+(DM+)に8〜24時間交換した。DM+は、液体Neurobasal(登録商標)−A培地(1×)、(Life Technologies, cat, No. 10888-022)、1×〜2× N2サプリメント(Life Technologies, cat, No.17502-048);1×〜2× B27サプリメント(Life Technologies, cat, No. 0080085-SA);1×〜2× インスリン−トランスフェリン−セレン−A(ITS-A, Life Technologies, cat, No. 51300-045);安定グルタミン(PAA, cat, No. M11-006);20ng/ml bFGF(PeproTech, cat, No. 100-18B)及び20ng/ml EGF(PeproTech, cat, No. AF-100-15)からなる。次に、培地をDMSOなしのDM+に交換し、35mmマトリゲルコーティングディッシュ上で1〜2日ごとに培地を交換して、細胞を集密になるまで10〜15日間成長させた。
骨格筋前駆細胞の誘導:35mmマトリゲルコーティングプレート(プレート1枚あたりhiPSC約2×106個)上でmTeSR(商標)1培地中に成長した成熟hiPSCコロニーを分化させた。1日目に、mTeSR(商標)1培地を、2%DMSO(Sigma-Aldrich, cat, No. D2438)を添加した分化培地+(DM+)に8〜24時間交換した。DM+は:液体Neurobasal(登録商標)A培地(1×)、(Life Technologies, cat, No. 10888-022)、1×〜2× N2サプリメント(Life Technologies, cat, No.17502-048);1×〜2× B27サプリメント(Life Technologies, cat, No. 0080085-SA);1×〜2× インスリン−トランスフェリン−セレン−A(ITS-A, Life Technologies, cat, No. 51300-045);安定グルタミン(PAA, cat, No. M11-006);10ng/ml〜20ng/ml bFGF(PeproTech, cat, No. 100-18B)及び10ng/ml〜20ng/ml EGF(PeproTech, cat, No. AF-100-15)からなる。次に、培地をDMSOなしのDM+に交換し、培地を1〜2日毎に交換して細胞を35mmマトリゲルコーティングディッシュ上で集密になるまで10〜15日間成長させた。
ニューロン前駆細胞及び骨格筋前駆細胞の誘導:35mmマトリゲルコーティングプレート上のmTeSR(商標)1培地中で成長した成熟hiPSCのコロニー(プレート1枚あたりhiPSC約2×106個)に分化を行った。1日目に、mTeSR(商標)1培地を2% DMSO(Sigma-Aldrich, cat, No. D2438)を添加した分化培地+(DM+)と8〜24時間交換した。DM+は:液体Neurobasal(登録商標)A培地(1×)、(Life Technologies, cat, No. 10888-022)、1×〜2× N2サプリメント(Life Technologies, cat, No.17502-048);1×〜2× B27サプリメント(Life Technologies, cat, No. 0080085-SA);1×〜2× インスリン−トランスフェリン−セレン−A(ITS-A, Life Technologies, cat, No. 51300-045);安定グルタミン(PAA, cat, No. M11-006);10ng/ml〜20ng/ml bFGF(PeproTech, cat, No. 100-18B)及び10ng/ml〜20ng/ml EGF(PeproTech, cat, No. AF-100-15)からなる。次に、培地をDMSOなしのDM+と交換し、1〜2日毎に培地を交換しながら細胞を35mmマトリゲルコーティングディッシュ上で10〜15日間、集密になるまで成長させた。
ニューロン前駆細胞及び筋前駆細胞への分化、それらの増殖を支援し、最終的に成熟機能性細胞へのそれらの分化を誘導するためにフィブロネクチン(R&D Systems, cat, No. 1918-FN-02M)及びラミニン(Invitrogen, cat, No. 23017-015)を使用する。
誘導の15日後に、細胞を機械的に破壊しラミニンコーティング2ウェルLabTek Permanoxチャンバースライド(Thermo Scientific, cat, No. 177429)に移植する。細胞を4%パラホルムアルデヒド中で20分間固定し、PBSで洗浄し、PBS 1×−0.1%トリトン中で20分間透過処理し、PBSで洗浄し、次に0.5% BSAを含有するブロッキング緩衝液に入れて室温で30分間インキュベートする。一次抗体とのインキュベーションは、4℃で一晩又は室温で2時間行った。インキュベーション後に、細胞をPBS中で洗浄し、0.5% BSAの存在下でAlexa fluorコンジュゲート型二次抗体(抗マウスAlexa Fluor 483(1/200)又は抗ウサギAlexa Fluor 555(1/1000))と共に1時間インキュベートした。核をDAPIで対比染色した。Axio Imager.Z2(Carl Zeiss Microscopy)を使用して画像を取得した。以下の一次抗体を使用した:マウス抗ネスチン(希釈1:500、Millipore, MAB5326);ウサギ抗NeuN(希釈1:100、Millipore, ABN78)、ウサギ抗チロシンヒドロキシラーゼ(希釈1:100、Millipore, AB152)、ウサギ抗OCT4(希釈1:100、Abcam, ab19857)、ウサギ抗MyoD(希釈1:100、Santa Cruz, SC304);ウサギ抗デスミン(希釈1:100、Abcam, Ab15200)、マウス抗MF20(希釈1:100)。抗マウスAlexa Fluor 483(希釈1:200、Ozyme 4408S)又は抗ウサギAlexa Fluor 555(希釈1:1000、Ozyme; 4413S)のいずれかを使用することによって視覚化を行った。
アキュターゼを用いて細胞を37℃で10分間処理し、N−培地でリンスする。細胞を計数し、1000rpmで5分間遠沈した。
Trizol(Invitrogen, Cat. No. 15596-026)を使用して全RNAを抽出した。Superscript IIキット及びオリゴdTを製造業者(Life Technologies)の説明書に従って使用して、全RNA 1μgの逆転写を42℃で50分間行い、続いて70℃で15分間不活性化した。Primer Blast及びPrimer 3を使用してプライマーを設計した。SYBRグリーンマスターミックスを使用してLightCycler 480(Roche)でリアルタイムPCR増幅を行った。標準化プロトコールを使用して全てのPCRを行い、Lightcycler 480ソフトウェアバージョン1.5.0.39(Roche)を用いてデータを解析した。標準曲線を使用する絶対定量及びRPL19標準遺伝子の発現に対する規準化により変化率を決定した:RPL19−F、ATC GAT CGC CAC ATG TAT CA(配列番号:1);RPL19−R,R:GCG TGC TTC CTT GGT CTT AG(配列番号:2)。
本発明者らは、ヒト多能性細胞(図4)からニューロン(図1)への分化を誘導するために簡単な手順を開発した。第1段階は、合成培地中に入れてマトリゲルコーティングプレート上で前駆細胞を予備分化及び増殖させることを要する。第2段階は、成熟ニューロン(図1)への最終分化のためにフィブロネクチン上の次にラミニンコーティングディッシュ上に播種することを要する。あるいは、特定の因子の存在下で特殊なニューロンの生成を達成することができる。
Claims (15)
- a)多能性幹細胞からニューロン細胞系列又は筋細胞系列に誘導され、フィブロネクチンでコーティングされた支持体上の、FGFファミリーのタンパク質のメンバー及びEGFを含む培地中で維持された細胞を提供する工程と、
b)ニューロンを得るためにラミニンでコーティングされた支持体か、筋管若しくは筋管とニューロンとの混合物を得るためにフィブロネクチンでコーティングされた支持体かのいずれかの上で、FGFファミリーのタンパク質のいかなるメンバーもEGFも含まない培地中で、前記誘導された細胞を培養する工程と
を含み、
胚様体を生成する工程を含まない、
ニューロン又は筋管を生成するための方法。 - 多能性幹細胞からニューロン細胞系列に誘導される細胞が、以下:
i. FGFファミリーのタンパク質のメンバー、EGF及びDMSOを含む培地中で多能性幹細胞を培養する工程;次に
ii. FGFファミリーのタンパク質のメンバー及びEGFを含み、DMSOを含まない培地中で前記細胞を培養する工程;並びに次に
iii. フィブロネクチンでコーティングされた支持体上の、FGFファミリーのタンパク質のメンバー及びEGFを含み、DMSOを含まない培地中で前記細胞を培養する工程
を用いて入手可能である、請求項1記載の方法。 - 前記多能性幹細胞が、人工多能性細胞(iPSC)又はES細胞である、請求項1又は2記載の方法。
- 前記iPSCが、胎児、小児又は成体の対象由来である、請求項3記載の方法。
- 前記iPSCが、胎児、小児又は成体の初代線維芽細胞由来である、請求項3又は4記載の方法。
- 培養する工程i)の持続時間が、8から24時間の間、特に持続時間16時間である、請求項2記載の方法。
- 培養する工程ii)の持続時間が、4から20日の間、特に15日である、請求項2又は6記載の方法。
- 工程iii)の持続時間が、少なくとも10日である、請求項2、6、及び7のいずれか一項記載の方法。
- 前記細胞が、工程a)と工程b)との間に凍結又は増殖されていてもよい、請求項1〜8のいずれか一項記載の方法。
- 工程a)で誘導された細胞が、ニューロンを得るために工程b)のラミニンでコーティングされた支持体上にそれらをプレーティングする前に酵素的又は機械的に破壊される、請求項1〜9のいずれか一項記載の方法。
- 工程a)で誘導された細胞が、ニューロンと筋管との混合物を得るために工程b)のフィブロネクチンでコーティングされた支持体上にそれらをプレーティングする前に剥がされる、請求項1〜9のいずれか一項記載の方法。
- 工程a)で誘導された細胞が、筋管を得るために工程b)のフィブロネクチンでコーティングされた支持体上にそれらをプレーティングする前に機械的に破壊される、請求項1〜9のいずれか一項記載の方法。
- 工程b)の持続時間が、少なくとも3日である、請求項1〜12のいずれか一項記載の方法。
- 工程b)の後に、ドーパミン作動性ニューロンなどの特殊なニューロンへのニューロンの終末分化のさらなる工程を含む、請求項1〜11のいずれか一項記載の方法。
- ドーパミン作動性ニューロンが、工程b)の後に得られた細胞を、FGF8及びSHHを含有する培地中で、特に持続時間48時間、培養することによって得られる、請求項14記載の方法。
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