JPWO2018097127A1 - 肝芽細胞から胆管上皮前駆細胞への段階的誘導方法 - Google Patents
肝芽細胞から胆管上皮前駆細胞への段階的誘導方法 Download PDFInfo
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Abstract
Description
肝芽細胞をTGFβおよびEGFを含む培地中で培養する工程を含む、胆管上皮前駆細胞を製造する方法を提供する。本願の方法によって、経時的にDP期様、RDP期様の胆管上皮前駆細胞を誘導することができる。
本願の方法は、胆管前駆細胞のDP期からRDP期への分化のメカニズムの解明、DPMに関連する疾患の病態解析、当該疾患のインビトロモデルの構築、当該疾患の治療のための候補物質のスクリーニング方法の構築などへの応用が期待できる。
HBMTMHepatocyte Basal Medium(Lonza)に下記を添加した培地が用いられる:
10% 10% KnockOutTM serum replacement (KSR: ThermoFisher Scientific)
10 ng/ml TGFβ2 (Peprotech)
25 ng/ml EGF (R&D)
HGF(肝細胞増殖因子)およびEGF(上皮成長因子)は市販品を用いれば良い。ヒト肝芽細胞を出発物質として用いる場合には、ヒト由来のHGFおよびEGFが好適に用いられる。
GSK3阻害剤としては、公知のもの、いずれを用いてもよく、例えばCHIR99021が例示される。
Notchシグナルのリガンドとしては、Delta様シグナル(Delta like Protein 1、Delta Like Protein 3, Delta Like Protein 4) 及びJaggedリガンド(Jagged-1 and Jagged-2)が例示される。Jagged-1が好適に用いられる。
10% KnockOutTM serum replacement (KSR: ThermoFisher Scientific)
20 ng/ml HGF (Peprotech)
50 ng/ml EGF (R&D)
50 ng/ml Jagged-1(R&D)
3μM CHIR99021 (StemRD)
培養温度は、以下に限定されないが、約30〜40℃、好ましくは約37℃であり、CO2含有空気の雰囲気下で培養が行われ、CO2濃度は、好ましくは約2〜5%である。
ヒトiPS細胞株23C27の樹立
ヒトAQP1の塩基配列が挿入されている大腸菌 (Bacterial Artificial Chromosome:Children's Hospital Oakland Research Instituteより入手)にGFP-PGK-Neoの塩基配列を挿入し、AQP1-GFPコンストラクトを作製した。コンストラクトの概略図を図1に示す。
AQP1-GFPコンストラクトをヒトiPS細胞585A1(京都大学iPS細胞研究所にて樹立)にエレクトロポレーションにより導入し、AQP1-GFPトランスジェニックヒトiPS細胞株 23C27を樹立した。このiPS細胞株から胆管上皮前駆細胞を誘導した。実施例の概略を図2に示す。
使用培地:
RPMI1640 (Nacalai Tesque) に下記添加:
1× B27 サプリメント (ThermoFisher Scientific)
1× ペニシリン/ストレプトマイシン (ThermoFisher Scientific)
100 ng/mL アクチビンA (R&D)
1-3 μM CHIR99021 (Day0: 3 μM, Day1-3: 1μM, Day3-5: 0 μM)(StemRD)
10 μM Y27632 (Day0: 10 μM, Day1-5: 0 μM)(Wako)
(1)で得た内胚葉細胞培養物(Day5)において、培地を下記に示す肝芽細胞誘導用培地へと交換した。肝芽細胞誘導用培地として、KnockOutTM DMEM (KODMEM; ThermoFisher Scientific) に下記を添加した培地を使用した:
10% KnockOutTM serum replacement (KSR: ThermoFisher Scientific)
1 mM L-グルタミン(ThermoFisher Scientific)
1% (vol/vol) 非必須アミノ酸(ThermoFisher Scientific)
1× ペニシリン/ストレプトマイシン
0.1 mM 2-メルカプトエタノール (ThermoFisher Scientific)
1% (vol/vol) DMSO (Sigma)
20 ng/ml BMP4 (Peprotech)
10 ng/ml FGF2 (Wako)
(2)において得られた肝芽細胞培養物(Day11)から胆管上皮前駆細胞を誘導した。胆管上皮分化誘導用培地としては、HBMTM Hepatocyte Basal Medium (Lonza) に下記を添加した培地を用いた:
10% KnockOutTM serum replacement (KSR; ThermoFisher Scientific)
10 ng/ml TGFβ2 (Peprotech)
25 ng/ml EGF (R&D)
3日間培養後(Day14)に一部の細胞を取り出し、常法により固定化し、胆管上皮前駆細胞マーカーについての免疫染色を行った。結果を図5−2に示す。得られた培養細胞はAPQ1が陰性であった。APQ1は胎生12〜16週程度で発現することから、Day14の培養細胞はこれらの期に満たないものであると推察される。また、胎生6〜8週程度までに認められるSOX9は広範囲に陽性であった。これらの結果より、Day14においては胎生8週程度のDuctal Plate期の胆管上皮前駆細胞と類似の遺伝子発現プロファイルを有していることが確認された。
Day18の細胞をさらに詳細な免疫染色に供した。結果を図5−4に示す。Day18の細胞はSOX9、CK19、AQP1およびCK7が陽性であり、胎生12週程度のRemodeling Ductal Plate期様の胆管上皮前駆細胞であることが確認された。
(3)においてDay18に得られた細胞集団からフローサイトメーターを用いてGFP陽性細胞を単離することによってAQP1陽性細胞とAQP1陰性細胞をそれぞれ単離した。胎生12〜20週程度の胆管上皮前駆細胞に発現することが知られているマーカー遺伝子の各細胞集団への発現について、PCRで確認した。コントロールとして、胎生20週の肝を用いて同様に各遺伝子の発現をPCRにて調べた。また一部のマーカー遺伝子については胆管上皮細胞を含む成人肝での発現についても同時に調べた。
さらに脳、膵臓、大腸および気管に特徴的なマーカー遺伝子として知られている遺伝子についても、その発現をPCRにて調べた。結果を図6−1から図6−3に示す。
本願の方法にて得られた胆管上皮前駆細胞は、GW12〜20程度の胆管上皮前駆細胞(remodeling ductal plate期)と同様の遺伝子発現プロファイルを示すことが確認された。一方で、他の臓器に特徴的な遺伝子についてはいずれも発現が認められなかった(図6−3)。
(2)で得られた肝芽細胞(Day11)、(3)で得られた胆管上皮前駆細胞 (Day14)を用いて、三次元培養を行った。
三次元スキャホールドの材料:
BDマトリゲルTM基底膜マトリックスグロースファクターリデュースト、10mlバイアル(BD 354230)
コラーゲンタイプI, Rat Tail, (ThermoFisher Scientific A1048301)
セルカルチャーインサートコンパニオンプレート(BD falcon 353504)
カルチャーインサート24 well用 1.0μm PET 透明 (FALCON #353104)
10% KnockOutTM serum replacement (KSR; ThermoFisher Scientific)
20 ng/ml HGF (Peprotech)
50 ng/ml EGF (R&D)
50 ng/ml Jagged-1(R&D)
3 μM CHIR99021 (StemRD)
使用試薬:
ローダミン123:カタログ番号R8004、Sigma-Aldrich
ベラパミル:カタログ番号V106-5MG、Sigma-Aldrich
ローダミン123は胆管上皮に発現するMDR1/p-glycoproteinとよばれるトランスポーターにより取り込まれる。べラパミルはMDR1/p-glycoproteinの阻害剤である。三次元管腔様構造を有する培養物がローダミン123を取り込むこと、およびベラパミルの存在下ではこのローダミンの細胞内への取り込みが顕著に抑えられることが確認された。この結果より、得られた三次元管腔様構造を有する培養物は、胆管としての生理機能を有していることが確認された。
Claims (14)
- 肝芽細胞を提供する工程、および
肝芽細胞をTGFβおよびEGFを含む培地で培養する工程を含む、胆管上皮前駆細胞の製造方法。 - TGFβがTGFβ2である、請求項1記載の方法。
- 誘導される胆管上皮前駆細胞が、CK19およびSOX9陽性、AQP1陰性の細胞である、請求項1または2記載の方法。
- 誘導される胆管上皮前駆細胞が、ductal plate期様胆管上皮前駆細胞である、請求項1〜3いずれかに記載の方法。
- 誘導される胆管上皮前駆細胞が、CK19、SOX9およびAQP1がいずれも陽性の細胞である、請求項1または2記載の方法。
- 誘導される胆管上皮前駆細胞が、remodeling ductal plate期様胆管上皮前駆細胞である、請求項1、2または5記載の方法。
- さらに多能性幹細胞から肝芽細胞を誘導する工程を含む、請求項1〜6いずれかに記載の方法。
- 多能性幹細胞がヒト由来の細胞である、請求項7記載の方法。
- 多能性幹細胞が、ヒトES細胞またはヒトiPS細胞である請求項8記載の方法。
- 請求項1〜9いずれかの方法により得られた、胆管上皮前駆細胞培養物。
- 胆管上皮前駆細胞がductal plate期様胆管上皮前駆細胞である、請求項10記載の培養物。
- 胆管上皮前駆細胞がremodeling ductal plate期様胆管上皮前駆細胞である、請求項10記載の培養物。
- 肝芽細胞または、胆管上皮前駆細胞を提供する工程、および
肝芽細胞または胆管上皮前駆細胞を、HGF、EGF、Notch阻害剤およびGSK3阻害剤を含む培地にて、三次元スキャホールド材の存在下で培養する工程を含む、胆管上皮前駆細胞三次元管腔様組織の構築方法。 - 肝芽細胞または、胆管上皮前駆細胞を提供する工程、および
肝芽細胞または胆管上皮前駆細胞を、HGF、EGF、Notch阻害剤およびGSK3阻害剤を含む培地にて、三次元スキャホールド材の存在下で培養する工程を含む方法により得られた、胆管上皮前駆細胞三次元管腔様組織。
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US20130189327A1 (en) * | 2010-07-29 | 2013-07-25 | Koninkijike Nederlandse Akademie Van Wetenschappen | Liver organoid, uses thereof and culture method for obtaining them |
JP2016517266A (ja) * | 2013-02-18 | 2016-06-16 | ユニバーシティー ヘルス ネットワーク | 多能性幹細胞から肝細胞及び胆管細胞を作製する方法 |
WO2016148216A1 (ja) * | 2015-03-18 | 2016-09-22 | 国立大学法人 東京大学 | 肝細胞及び肝非実質細胞、並びにそれらの調製方法 |
WO2016207621A1 (en) * | 2015-06-22 | 2016-12-29 | Cambridge Enterprise Limited | In vitro production of cholangiocytes |
WO2017119512A1 (ja) * | 2016-01-08 | 2017-07-13 | 国立研究開発法人国立がん研究センター | 低分子化合物による成熟肝細胞からの肝幹/前駆細胞の作製方法 |
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US20130189327A1 (en) * | 2010-07-29 | 2013-07-25 | Koninkijike Nederlandse Akademie Van Wetenschappen | Liver organoid, uses thereof and culture method for obtaining them |
JP2016517266A (ja) * | 2013-02-18 | 2016-06-16 | ユニバーシティー ヘルス ネットワーク | 多能性幹細胞から肝細胞及び胆管細胞を作製する方法 |
WO2016148216A1 (ja) * | 2015-03-18 | 2016-09-22 | 国立大学法人 東京大学 | 肝細胞及び肝非実質細胞、並びにそれらの調製方法 |
WO2016207621A1 (en) * | 2015-06-22 | 2016-12-29 | Cambridge Enterprise Limited | In vitro production of cholangiocytes |
WO2017119512A1 (ja) * | 2016-01-08 | 2017-07-13 | 国立研究開発法人国立がん研究センター | 低分子化合物による成熟肝細胞からの肝幹/前駆細胞の作製方法 |
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ORGANOGENESIS, vol. 4, JPN6018005342, 2008, pages 92 - 99, ISSN: 0004742446 * |
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