JP6599468B2 - 小分子化合物を利用したヒト線維芽細胞を神経幹細胞に直接転換する方法 - Google Patents
小分子化合物を利用したヒト線維芽細胞を神経幹細胞に直接転換する方法 Download PDFInfo
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Description
1−1:小分子化合物の機能確認
ヒト線維芽細胞を神経幹細胞に誘導するため、リプログラミング(Reprogramming)に関連した様々な小分子化合物中13種を選択的に選別して使用した(表1)。
最も効率的な神経幹細胞製作条件のために、実施例1−1で確認した8個の小分子物質を一度に添加した条件から小分子物質1個ずつを引いた様々な条件の培養液を利用して線維芽細胞が神経幹細胞に変化するか否かを確認した。ヒト線維芽細胞1×105個を60mm dishに準備して翌日から下記の表2の様々な条件の培養液を2〜3日毎に取り替えながら細胞の形態学的変化を確認した。また、継代培養をしながら得られた細胞を介して様々な遺伝子の発現パターンの変化を観察した。
ヒト線維芽細胞を神経幹細胞に変換するのに必要とする小分子物質の組み合わせが定まるにつれ、安定的に誘導と培養する条件を確立しよう試みた。また、神経幹細胞に誘導される効率を確認するために、最小4個の小分子化合物が添加された培養液を利用して比較した。まず、1×105個の線維芽細胞を準備した後、DMEM/F12、N2、B27、bFGF、EGFで構成されたneurobasal mediumに4種(Thiazovivin、Valproic acid、Purmorphamine及びA8301)、6種(Thiazovivin、Valproic acid、Purmorphamine、A8301、SB431542及びCHIR99021)、8種(Thiazovivin、Valproic acid、Purmorphamine、A8301、SB431542、CHIR99021、Deazaneplanocin A及び5−AZA)の小分子化合物がそれぞれ添加されている3つの条件の培養液を処理した。
DMEM/F12+N2B27に6個の小分子化合物で誘導及び維持したヒト神経幹細胞が神経幹細胞の一般的な特性を示すのか否かを確認した。まず、神経幹細胞が神経幹細胞のマーカーを発現するか否かを確認しようとPCRとimmunocytochemistry(ICC)を進めた。その結果、免疫細胞化学(ICC)染色を介して神経幹細胞がネスチンを発現するのを確認して、PCRを介して神経幹細胞マーカー(ネスチン、sox1、musashi 1)の遺伝子発現がヒト胚性幹細胞で由来した神経幹細胞と似たような発現程度を示すことを確認した(図4)。
実施例1〜3を介して小分子物質を利用してヒト線維芽細胞から神経幹細胞を誘導することが成功的に行われたことを確認した。より具体的に、神経幹細胞の特性及び利用の可能性を確認するために、in vitro上での分化能力を確認した。まず、3神経細胞であるアストロサイト(astrocyte)、オリゴデントロサイト(oligodendrocyte)及びニューロン(neuron)に分化を進めた。
その結果、小分子化合物によって誘導された神経幹細胞は成功的に3神経細胞に分化されることを確認した(図9)。
このようなヒト線維芽細胞を利用したヒト神経幹細胞への誘導、増殖及び種々の機能性神経細胞への分化能力は、ヒトの脳疾患治療のための細胞治療剤への活用の可能性が非常に高いことを示している。
実施例3及び4では、小分子物質を利用してヒト線維芽細胞から誘導された神経幹細胞が成功的に3神経細胞に分化が可能であることをin vitro上で確認した。
Claims (11)
- チアゾビビン(Thiazovivin)、バルプロ酸(Valproic acid)、パルモルファミン(Purmorphamine)、A8301、SB431542、CHIR99021、DZNep(Deazaneplanocin A)、及び5−AZAを含む培地でヒト線維芽細胞を培養する段階を含む神経幹細胞の製造方法。
- 前記培地は、PD0325901、アスコルビン酸(Ascorbic acid)、PS48、ホルスコリン(Forskolin)及びトラニルシプロミン(Tranylcypromine)で構成された群から選択された1種以上の小分子化合物をさらに含むことを特徴とする請求項1に記載の神経幹細胞の製造方法。
- 前記培地は、N2、B27、bFGF及びEGFが含まれたDMEM/F12であることを特徴とする請求項1に記載の神経幹細胞の製造方法。
- 前記培養は、10〜15日間行われることを特徴とする請求項1に記載の神経幹細胞の製造方法。
- 前記ヒト線維芽細胞を継代培養した後、浮遊培養してスフィアを形成する段階;及び形成された前記スフィアを付着培養した後、再び浮遊培養する段階をさらに含むこと特徴とする請求項1に記載の神経幹細胞の製造方法。
- 前記浮遊培養及び付着培養は、それぞれ7〜10日間行われることを特徴とする請求項5に記載の神経幹細胞の製造方法。
- 形成された前記スフィアを付着培養した後、再び浮遊培養する段階を2〜4回繰り返すことを特徴とする請求項5に記載の神経幹細胞の製造方法。
- 前記神経幹細胞は、ネスチン(nestin)、sox1またはmusashi 1を発現することを特徴とする請求項1に記載の神経幹細胞の製造方法。
- 前記神経幹細胞は、星状細胞(astocyte)、乏突起膠細胞(oligodendrocyte)、ニューロン(neuron)、ドーパミン神経細胞、GABA性神経細胞、運動神経細胞およびコリン作動性ニューロンで構成された群から選択されるいずれか一つ以上に分化することを特徴とする請求項1に記載の神経幹細胞の製造方法。
- 前記神経幹細胞は、染色体安定性を維持させることを特徴とする請求項1に記載の神経幹細胞の製造方法。
- 前記神経幹細胞は、10継代以上未分化状態を維持することを特徴とする請求項1に記載の神経幹細胞の製造方法。
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