JPWO2019131938A1 - 機能的肝前駆細胞もしくは肝細胞または機能的小腸上皮前駆細胞もしくは小腸上皮細胞を調製する方法 - Google Patents
機能的肝前駆細胞もしくは肝細胞または機能的小腸上皮前駆細胞もしくは小腸上皮細胞を調製する方法 Download PDFInfo
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Abstract
Description
劇症肝炎患者の線維芽細胞から、以下の手順によりiPS細胞を作製した。劇症肝炎患者(男性・0歳7ヶ月)から生検により得られた皮膚を培養し、線維芽細胞を得た。得られた線維芽細胞に、Oct3/4、Sox2、Klf4およびc−Mycのヒト遺伝子を、センダイウイルスベクターを用いて導入した。その後、2週間培養し、ドーム状のコロニーの出現を確認した。出現したコロニーを単離し、MEFフィーダー細胞上に移し、hiPS培地(組成は下記)中で継代培養した。
(2−1.胚様体(EB)の形成)
上記iPSC−K細胞が約80%コンフルエントになるまで培養した。培地を除去し、PBSにより洗浄した後、StemPro Accutase(Thermo Fisher Scientific)を添加し、10〜20分間、37℃、5%CO2条件下でインキュベートした。ディッシュを軽く叩いてコロニーを剥がした後、hiPS培地を加え、懸濁した。得られた細胞懸濁液をゼラチンコートされたディッシュに移し、15分間、37℃、5%CO2条件下でインキュベートすることにより、フィーダー細胞を除去した。上清を遠心し、回収されたiPSC−K細胞をEB培地(組成は下記)に懸濁した。浮遊培養用の96ウェルプレート(Thermo Fisher Scientific、174929)に、1×106細胞/ウェルの濃度でiPSC−K細胞を播種し、37℃、5%CO2条件下で10日間培養し、胚様体(以下、「EB」と記載する)を形成させた。
EBを回収し、XF32培地(Uchida,H.et al.,JCI Insight.,2017;2(1):e86492,doi:10.1172/jci.insight.86492)に懸濁した。コラーゲンコートされた24ウェルプレートに20EBs/ウェルの濃度でEBを播種し、37℃、5%CO2にて35日間、接着培養を行うことにより肝前駆細胞へと分化誘導した。その後、3μg/mlのピューロマイシン(Wako、#160−23151)を添加した改変SCM−6F8培地(Wang,X.et al.,Nature,2015;522(7555):173−8.,doi:10.1038/nature14484.,Epub 2015 Jun 3)を用いて、さらに2日間培養した。
続いて、ヒトテロメラーゼ逆転写酵素(TERT)遺伝子、変異ヒトCDK4(CDK4R24C)遺伝子、およびヒトサイクリンD1遺伝子を、上記により得られた機能的肝前駆細胞に導入し、不死化した。上記遺伝子の導入は、それぞれ、レンチウイルスベクターCSII−CMV−hTERT、CSII−TRE−Tight−hCDK4R24C、およびCSII−TRE−Tight−cyclin D1を用いて、PLoS One,2012;7(1):e29677.,doi:10.1371/journal.pone.0029677.,Epub 2012 Jan 19.に記載の手順にしたがって行った。得られた不死化機能的肝前駆細胞は、「HepaSM」と命名され、国際寄託当局である独立行政法人製品評価技術基盤機構特許微生物寄託センターに、受託番号BP−02591として寄託された(寄託日:2017年12月6日、受領番号AP−02591)。
HepaSM細胞を分化誘導して得られた細胞について、ヘマトキシリン・エオシン染色および肝細胞マーカーによる免疫染色を行った。肝細胞マーカーによる免疫染色は、以下の手順により行った。培地を除去し、4%パラホルムアルデヒド溶液を加え、4℃で10分間、細胞を固定した。溶液を除去後、0.1%Triron−X(ナカライテスク)溶液を加え、室温で10分間細胞を透過処理した。溶液を除去後、Protein Block Serum−Free Ready−to−use(Dako,X0909)を加え、室温で30分間ブロッキング処理を行った。一次抗体として、抗CYP3A4抗体(C−17)(SANTA CLUZ BIOTECHNOLOGY,sc−27639)(1/1000希釈)、抗アルブミン抗体(CEDARLANE,CLFAG2140)(1/50希釈)、または抗α−フェトプロテイン(AFP)抗体(B&D SYSTEM,MAB1368)(1/100希釈)を用い、4℃で一晩反応させた。二次抗体には、Rabbit anti−Goat IgG(H+L)Cross−Adsorbed Secondary Antibody,Alexa Fluor 488(Thermo Fisher Scientific,A11078)、GFP Tag Polyclonal Antibody,Alexa Fluor 488(Thermo Fisher Scientific,A21311)、またはGoat anti−Mouse IgG1 Cross−Adsorbed Secondary Antibody,Alexa Fluor 546(Thermo Fisher Scientific,A21123)(いずれも1/1000希釈)をそれぞれ用い、室温で30分間反応させた。
HepaSM細胞を分化誘導して得られた肝細胞について、シトクロムP450の発現に基づく薬物代謝酵素誘導試験を行った(Khuu,D.N.et al.;In Vitro Differentiated Adult Human Liver Progenitor Cells Display Mature Hepatic Metabolic Functions: A Potential Tool for In Vitro Pharmacotoxicological Testing,Cell Transplantation.,2011;20(2):287−302)。リファンピシン(溶媒:DMSO)を、終濃度20μMになるように改変SCM−6F8培地に加え、2日間培養し、その後、上記3と同様の手順により、CYP1A2、CYP2B6およびCYP3A4の発現量をqRT−PCRにより定量した。また、リファンピシンに代えてDMSOを同量添加した培地を用いて同様にして調製した細胞(図中、「RFP(−)」と表記)を対照とした。
HepaSM細胞を分化誘導して得られた肝細胞の増殖能を、増殖細胞マーカーであるKi67およびPCNAの発現に基づいて評価した。細胞をパラフィン包埋することにより固定した後、脱パラフィン処理し、PBSで洗浄した。その後、2.5%ヤギ血清/PBSを加え、室温で30分間ブロッキング処理を行った。一次抗体として、抗Ki67抗体(DAKO,PC10)または抗PCNA抗体(DAKO,MIB−1)(いずれも1/200希釈)を用い、室温で1時間反応させた。二次抗体反応およびDAB発色は、ImmPRESS Anti−Mouse IgG Kit(Vector Labs)を用いて行った。水で洗浄後、ヘマトキシリン・エオシン染色を行い、得られた標本を顕微鏡観察した。
ピューロマイシンに代えて各種抗生物質を用いた以外は上記(2−1)および(2−2)と同様の手順により、iPSC−K細胞から機能的肝前駆細胞を調製した。用いた抗生物質およびその濃度、ならびに培養日数を以下に示す。
以下の手順により、ヒトES細胞であるSEES5細胞(Akutsu,H.et al.,Regen.Ther.,2015;1:18−29,doi:10.1016/j.reth.2014.12.004)から機能的肝前駆細胞を調製した。hiPS培地に代えてEssential8培地(Gibco)を用いた以外は上記(2−1)と同様の手順により、EBを形成させた。EBを回収し、XF32培地に懸濁した。コラーゲンコートされた24ウェルプレートに20EBs/ウェルの濃度でEBを播種し、37℃、5%CO2にて10日間、接着培養を行った。その後、HD培地(XF32培地からbFGFのみを抜いた組成からなる)に交換して10日間培養し、さらに改変SCM−6F8培地に交換して7日間培養した。その後、各種抗生物質を添加した改変SCM−6F8培地に交換した。用いた抗生物質およびその濃度、ならびに培養日数を以下に示す。
iPSC−K細胞に代えてiPSC−O細胞を用いた以外は上記(2−1)および(2−2)と同様の手順によりiPSC−O細胞から調製されピューロマイシンにより選択された機能的肝前駆細胞、ならびに、上記7においてSEES5細胞から調製されピューロマイシンにより選択された機能的肝前駆細胞について、以下の手順によりアンモニアによるさらなる選択培養を行った。iPSC−O細胞から調製された機能的肝前駆細胞またはSEES5細胞から調製された機能的肝前駆細胞の培養物(10cmディッシュ)から培地を除去し、5mlのD−PBS(Gibco)により洗浄した。その後、それぞれの培養物に対し、5mg/mlのアンモニアを添加した改変SCM−6F8培地または8mg/mlのアンモニアを添加した改変SCM−6F8培地を10ml加え、37℃、5%CO2条件下で2日間培養した。その後、培地を除去し、5mlのD−PBS(Gibco)により洗浄し、10mlの新しい改変SCM−6F8培地を添加した。
Uchida,H.et al.,JCI Insight.,2017;2(1):e86492に記載の手順に従って、月経血由来のiPS細胞であるEdom−iPS細胞(PLoS Genet.,2011;7(5):e1002085,doi.org/10.1371/journal.pgen.1002085)から腸オルガノイドを調製した。3ヶ月経過した腸オルガノイドを、2mg/mlまたは400mg/mlのピューロマイシン存在下で2日間培養した。
Claims (22)
- 機能的肝前駆細胞もしくは機能的肝細胞または機能的小腸上皮前駆細胞もしくは機能的小腸上皮細胞を調製する方法であって、肝前駆細胞もしくは肝細胞または小腸上皮前駆細胞もしくは小腸上皮細胞を含む単離細胞集団を抗生物質の存在下で培養するステップを含む、方法。
- 前記単離細胞集団が肝組織または小腸上皮組織由来の初代培養物である、請求項1に記載の方法。
- 前記単離細胞集団が幹細胞から分化誘導されたものである、請求項1に記載の方法。
- 前記幹細胞が胚性幹(ES)細胞である、請求項3に記載の方法。
- 前記幹細胞が人工多能性幹(iPS)細胞である、請求項3に記載の方法。
- 前記iPS細胞が健常者由来のものである、請求項5に記載の方法。
- 前記iPS細胞が薬物性肝障害患者または薬物性小腸障害患者由来のものである、請求項5に記載の方法。
- 前記抗生物質が、ピューロマイシン、ブラストサイジンS、G418、ハイグロマイシン、フレオマイシンおよびフレオマイシンD1からなる群から選択される、請求項1〜7のいずれか1項に記載の方法。
- 前記抗生物質が0.1〜100μg/mlのピューロマイシンである、請求項1〜8のいずれか1項に記載の方法。
- 前記培養ステップが肝分化誘導因子の存在下で行われる、請求項1〜9のいずれか1項に記載の方法。
- 前記肝分化誘導因子が、サイトカイン、細胞増殖因子、ROCK阻害剤、MAPK阻害剤、ALK阻害剤および細胞外基質からなる群から選択される、請求項10に記載の方法。
- 前記単離細胞集団をアンモニアの存在下で培養するステップをさらに含む、請求項1〜11のいずれか1項に記載の方法。
- 請求項1〜12のいずれか1項に記載の方法により得られる、機能的肝前駆細胞または機能的肝細胞。
- 請求項1〜12のいずれか1項に記載の方法により得られる、機能的小腸上皮前駆細胞または機能的小腸上皮細胞。
- 機能的肝前駆細胞を含む実質的に均一な単離細胞集団であって、前記機能的肝前駆細胞におけるCYP3A4の発現量が、HepaRG(登録商標)細胞株におけるその発現量と比較して少なくとも5倍増大している、単離細胞集団。
- 前記機能的肝前駆細胞が、α−フェトプロテイン、アルブミン、CYP1A2およびCYP2B6からなる群から選択される少なくとも1つを、HepaRG(登録商標)細胞株よりも高いレベルで発現している、請求項15に記載の単離細胞集団。
- 前記機能的肝前駆細胞が、CD44陽性および/またはEpCAM陽性である、請求項15または16に記載の単離細胞集団。
- 不死化されている、請求項15〜17のいずれか1項に記載の単離細胞集団。
- 独立行政法人製品評価技術基盤機構特許微生物寄託センターに受託番号BP−02591および/またはBP−02592として寄託された、機能的肝前駆細胞。
- 肝機能障害を有する対象を治療する方法であって、請求項15〜18のいずれか1項に記載の単離細胞集団を前記対象に投与するステップを含む、方法。
- 請求項15〜18のいずれか1項に記載の単離細胞集団を含んでなる、肝機能障害を治療するための医薬組成物。
- (1)請求項15〜18のいずれか1項に記載の単離細胞集団または請求項19に記載の機能的肝前駆細胞を分化誘導して得られた機能的肝細胞と試験化合物を接触させるステップと、
(2)前記機能的肝細胞の障害の程度を解析するステップと
を含む、試験化合物の毒性評価方法。
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