JP6983907B2 - 奇形腫の形成が抑制された多分化能性幹細胞由来の神経前駆体球の製造方法 - Google Patents
奇形腫の形成が抑制された多分化能性幹細胞由来の神経前駆体球の製造方法 Download PDFInfo
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Description
また、本発明は、前記の方法で製造された神経前駆体球を提供する。
さらに、本発明は、前記の神経前駆体球を分化させる段階と、を含む、神経細胞の分化方法を提供する。
本発明は、1)神経前駆体球を単一細胞化する段階と、2)前記の段階1)の単一細胞化された神経前駆体球を再凝集させる段階と、及び3)前記の段階2)の再凝集された神経前駆体球を回収する段階と、を含む奇形腫の形成が抑制された神経前駆体球の製造方法を提供する。本発明に係る製造方法は、図1のBに、従来の製造方法は、図1のAに簡略に示した。
本明細書にて用いられる用語、「神経前駆体球」とは、ヒト胚性幹細胞または誘導多分化能性幹細胞のような多分化能性幹細胞から作られた神経前駆細胞または神経組織から分離された神経前駆細胞が球状に凝集されたものを意味する。
前記神経前駆体球は、上述したような製造方法によって製造することができる。具体的には、前記の方法は、1)神経前駆体球を単一細胞化する段階と、2)前記の段階1)の単一細胞化された神経前駆体球を再凝集させる段階と、及び3)前記の段階2)の再凝集された神経前駆体球を回収する段階と、を含むことができる。
前記神経前駆体球は、上述したような特徴を含むことができる。また、前記の分化において、bFGFの代わりに添加されるB27は、全分化用培地の1〜3%(v/v)、より具体的には、1.5%〜2.5%(v/v)の量で添加されることができる。前記B27の添加量が1%(v/v)未満であれば、細胞の分化効率が低下することができ、3%(v/v)を超えると、過剰な分化及び増殖により細胞培養の維持に問題が生じることがある。本発明の一実施例によれば、前記B27の添加量は、2%(v/v)であってもよい。
但し、下記の実施例は、本発明を例示するものであり、本発明の内容がこれらによって限定されるものではない。
本発明に係る多分化能性幹細胞から形成された神経前駆体球の切片から神経前駆体球の再形成過程を通して神経前駆細胞のみを純粋に分離した。
まず、神経前駆細胞の基礎培地は、490mlのDMEM(F12)に5mlの0.1mM NEAA(non essential amino acids)と5mlの2mM P/S(penicillin/streptomycin)を添加して製造した。製造された培地をよく混ぜた後、0.22μmのポアサイズを有するフィルタを用いて、真空ポンプで濾過し、これを50mlのチューブに49mlずつ分注した。前記チューブをパラフィンフィルムで密封し、4℃で保管した。培地の量が12ml未満であれば、15mlチューブに、12ml以上であれば、50mlチューブに入れて保管した。
神経前駆細胞を培養するための皿の大きさは、神経前駆体球の数に応じて決まる。本実施例では、直径35mmのシャーレ当たり、切片化された10個の神経前駆体球を用いた(1凝集物/cm2)。シャーレは使用する前に、1mlに対し20倍希釈されたマトリゲルを入れて、1時間以上、常温保管して用いた。
<2−1>神経前駆体球の神経細胞への分化能を確認
まず、実験に用いられる神経前駆体球を免疫染色して、前記細胞が神経細胞への分化能があることを確認した。
また、前記神経前駆体球が神経前駆細胞マーカーを発現するかどうかを免疫染色で確認した。神経前駆体球は、凍結切片化した後、カバーガラスに付着して免疫染色し、免疫染色方法は、前記実施例<2−1>と同様の条件及び方法で行われた。このとき、免疫学的染色のためにrabbit anti−Musashi antibody(1:100、Chemicon)、rabbit anti−Sox1 antibody(1:200、Milipore)、mouse anti−human Nestin antibody(1:200、Chemicon)及びmouse anti−Pax6 antibody(1:500、Novus Biologicals)を1次抗体として使用し、2次抗体としては、Alexa Fluor 488 donkey anti−mouse IgGとAlexa Fluor594 donkey anti−rabbit IgG(1:200、Molecular Probes、USA)を用いて実験を行った。
<3−1>神経前駆体球の切片化(schizolysis)
まず、神経前駆体球を100μm3の体積を超えない大きさに、微細切片ツールを用いて切片化した。具体的には、ガラスパスツールピペット(pipette)を細く引き伸ばしたものを使用して、神経前駆体球を切片化した。切片化された神経前駆体球は、<実施例1>の神経前駆細胞の培養用培地及びマートリゲルコーティングされたシャーレを使用して、37℃、5%のCO2の条件で12時間の間、付着培養した。
切片化された神経前駆体球の単一細胞化
切片化された神経前駆体球を次のような方法で単一細胞化させた。
まず、<3−1>で培養された神経前駆体球の培地を除去し、PBS(phosphate buffered saline)で軽く洗浄した。PBSを除去し、0.2ml/cm2のアクターゼ(accutase)を添加した後、37℃、5%のCO2の条件で40〜60分間反応させ、付着された神経前駆体球の切片を、単一細胞化させた。これらを遠心分離して集めた後、アクターゼを除去し、神経前駆細胞の培養用培地で洗浄し、1mlの培養用培地を入れた後、計数した。
次に、神経前駆体球を切片化し、単一細胞化させることが、細胞の生存率及び回収率に影響を与えるか確認した。
前記<3−2>にて単一細胞化された細胞を再び自発的に再凝集させるため、次のように処理した。
前記<3−3>にて再凝集された神経前駆体球の直径が50μm〜100μmになったとき、これを回収して、神経細胞への分化を誘導した。
再凝集された神経前駆体球の細胞が、神経前駆細胞の特性をそのまま維持するのかを確認した。
まず、アクターゼを処理して、神経前駆体球を単一細胞化させた後、これをPBSで洗浄し、固定液で固定し、免疫染色を行った。未分化細胞の存在有無は、未分化の多分化能性幹細胞としてよく知られているマーカーであるSSEA−4及びTRA−61タンパク質の染色により確認した。神経前駆細胞の純度は、神経前駆細胞マーカーであるMusashi及びPax6タンパク質を用いて確認した。対照群としては、ヒト胚性幹細胞(ES)を用い、既存の神経前駆体群(SNM)と再形成された神経前駆体球(Reformed SNM)を一緒に比較した。
ヒト胚性幹細胞、胚体、神経前駆体球及び再凝集された神経前駆体球それぞれの遺伝子発現の変化をRT−PCRで確認した。遺伝子の発現は、未分化の幹細胞から主に発現されるOct4及びナノグ(Nanog)、神経前駆細胞から主に発現されるSox1、Pax6及びネスチン(nestin)は、初期の神経細胞から主に発現されるNF−Mをマーカーとして確認した。
再凝集された神経前駆体球の分化能が維持されることを確認するために、免疫組織学的染色を行った。
Claims (12)
- 1)神経前駆体球を切片化する段階と、
2)前記切片化された神経前駆体球を、神経前駆細胞の培養用培地を含む培養皿中で付着培養する段階と、
3)細胞分離用酵素を添加することにより前記付着培養した神経前駆体球を単一細胞化して、単一細胞を製造する段階と、
4)前記段階3)の単一細胞を、基質がコーティングされていない培養皿中で付着培養する段階と、
5)前記培養皿の底に付着した凝集物を繰り返し浮遊させて前記単一細胞の自発的な再凝集を誘導する段階と、
6)前記段階5)の再凝集された神経前駆体球を回収する段階と、
を含む奇形腫の形成が抑制された高純度の神経前駆体球の製造方法。 - 前記神経前駆細胞の培養用培地が、細胞の培養用培地としてN−2添加物(登録商標)、bFGFまたはそれらの混合物を含む、請求項1に記載の神経前駆体球の製造方法。
- 前記N−2添加剤(登録商標)は、前記培養用培地の総体積に対して0.5〜2.0%(v/v)の量で添加される、請求項2に記載の神経前駆体球の製造方法。
- 前記bFGFは、前記培養用培地の総質量に対して0.0002〜0.001%(w/v)の量で添加される、請求項2に記載の神経前駆体球の製造方法。
- 前記の段階2)が基質がコーティングされた培養皿で行われる、請求項1に記載の神経前駆体球の製造方法。
- 前記の基質がラミニン、L−オルチニン、マートリゲル及びCeLLstartからなる群から選択されるいずれか一つ以上を含む、請求項5に記載の神経前駆体球の製造方法。
- 前記の段階5)の再凝集された神経前駆体球が直径10〜80μmの大きさである、請求項1に記載の神経前駆体球の製造方法。
- 請求項1の方法で製造された、奇形腫の形成が抑制された神経前駆体球。
- 1)神経前駆体球を切片化する段階と、
2)前記切片化された神経前駆体球を、神経前駆細胞の培養用培地を含む培養皿中で付着培養する段階と、
3)細胞分離用酵素を添加することにより前記付着培養した神経前駆体球を単一細胞化して、単一細胞を製造する段階と、
4)前記段階3)の単一細胞を、基質がコーティングされていない培養皿中で付着培養する段階と、
5)前記培養皿の底に付着した凝集物を繰り返し浮遊させて前記単一細胞の自発的な再凝集を誘導する段階と、
6)前記段階5)の再凝集された神経前駆体球を回収する段階と、
7)前記の段階6)の回収した神経前駆体球を神経細胞に分化させる段階と、を含む、神経前駆体球の神経細胞への分化方法。 - 前記分化がbFGFを添加することなくB27(登録商標)が添加された培地で行われる請求項9に記載の神経細胞の分化方法。
- 前記B27(登録商標)は、前記培地の総体積に対して1〜3%(v/v)の量で添加される請求項10に記載の神経細胞の分化方法。
- 前記神経前駆体球が直径500μm以上で継代される請求項9に記載の神経細胞の分化方法。
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