JP7463326B2 - 胃底部組織のインビトロでの製造のための方法及び当該方法と関連した組成物 - Google Patents
胃底部組織のインビトロでの製造のための方法及び当該方法と関連した組成物 Download PDFInfo
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Description
本出願は、2016年5月5日出願の米国仮特許出願第62/332,194号に対する優先権及び利益を主張し、その内容がすべて参照により組み込まれる。
本発明は、All 16491及びDK092456の下での政府の支援を用いて行われた。当該政府は、本発明におけるある特定の権利を有する。
いくつかの実施形態において、重要なステップとは、多能性である、または多能性となるよう誘導することのできる幹細胞を得ることである。いくつかの実施形態において、多能性幹細胞は、胚性幹細胞に由来し、これが順に、初期哺乳類胚の多能性細胞に由来し、制限されない未分化の増殖をインビトロですることができる。胚性幹細胞とは、初期胚である胚盤胞の内部細胞塊に由来する多能性幹細胞である。胚盤胞から胚性幹細胞を得るための方法は、当該技術分野で周知である。ヒト胚性幹細胞H9(H9-hESC)は、本出願において説明される例示的な実施形態において使用されるが、本明細書で説明される方法及びシステムが、いかなる幹細胞に対しても応用可能であることは、当業者によって理解されるであろう。
いくつかの実施形態において、iPSCは、ある特定の幹細胞関連遺伝子を成体線維芽細胞などの非多能性細胞へとトランスフェクションすることによって得られる。トランスフェクションは典型的には、レトロウイルスなどのウイルスベクターを通じて達成される。トランスフェクトした遺伝子は、マスター転写調節因子Oct-3/4(Pouf51)及びSox2を含むが、他の遺伝子が誘導効率を高めることが示唆されている。3~4週間後、少数のトランスフェクトした細胞は、多能性幹細胞と形態学的に及び生化学的に類似することとなり始め、典型的には形態学的選別、倍加時間を通じて、またはレポーター遺伝子及び抗生物質による選別を通じて単離される。本明細書で使用する場合、iPSCは、マウスにおける第1世代iPSC、第2世代iPSC、及びヒト人工多能性幹細胞を含むが、これらに限定されない。いくつかの実施形態において、レトロウイルスシステムを使用して、4つの中心となる遺伝子を用いてヒト線維芽細胞を多能性幹細胞へと形質転換する。すなわち、Oct3/4、Sox2、Klf4、及びc-Mycである。代替的な実施形態において、レンチウイルスシステムを使用して、体細胞をOCT4、SOX2、NANOG、及びLIN28で形質転換する。iPSCにおいて発現が誘導される遺伝子は、Oct-3/4(例えば、Pou5fl)、Sox遺伝子ファミリーのある特定のメンバー(例えば、Sox1、Sox2、Sox3、及びSox15)、Klfファミリーのある特定のメンバー(例えば、Klf1、Klf2、Klf4、及びKlf5)、Mycファミリーのある特定のメンバー(例えば、C-myc、L-myc、及びN-myc)、Nanog、及びLIN28を含むが、これらに限定されない。
胚発生中の胃底部特異性を調節する経路の研究に助力するために、発明者は、マウス胚を分析して、予定胃底部と予定前庭と予定前胃とを区別することのできる分子マーカーを識別した。14.5日胚で、発明者は、Sox2が前腸器官系譜前部において発現したのに対し、Gata4が腺性胃上皮へと制限されることを発見した。Gata4+部内で、Pdx1は、予定前庭領域に特異的であったので(図6のa)、胚性胃底部は、Sox2+Gata+Pdx1-であると考えられる。さらに、発明者は、公開されたマイクロアレイデータセット(GSM326648-GSM32665012及びGSM80809-GMS8081613)及び14.5日胚前腸の摘出した領域を分析して、転写因子Irx2、Irx3、及びIrx5の発現が、前庭と比較して胚性胃底部において10倍超多量であることを実証し(図6のb、c)、このことは、当該転写因子の発現が、腺性胃上皮の領域間をさらに区別することができることを示した。
発明者は次に、発生中のヒト胃の胃底部-前庭パターンを確立するうえでのWnt/βカテニンシグナル伝達の役割を研究した。胃の分化の初期をモデル化するために、発明者は、高い信頼度で初期胃発生の正常期を再現する、hPSCを前庭様胃オルガノイドへと分化させるための既に説明したプロトコルを用いて出発した7。3次元後部前腸スフェロイド(SOX2+HNF1β+)を用いて出発して、発明者は、Wnt/βカテニンシグナル伝達の刺激が、胃の特化期の間に前庭(SOX2+GATA+PDX1+)よりもむしろ胃底部(SOX2+GATA+PDX1-)の系譜へと後部前腸上皮を定向化するかどうかを検査した(図2のa)。実際、βカテニンをGSK3阻害因子CHIR99021(CHIR)で3日間活性化した結果、9日後にPDX1のほぼ完全な抑止を生じるとともに、IRX2、IRX3、及びIRX5の発現を有意に増加させた(図2のb~c)。重要なことに、SOX2及びGATA4のレベルは、CHIR処理によって影響されず、スフェロイドがそれらの胃の同一性を保有していることを確証した。したがって、CHIR曝露は結果的に、SOX2+GATA+PDX1上皮の形成を生じるとともに、予定胃底部上皮と一致した兆候であるIRX発現の増加が生じた。
分化した前庭胃細胞タイプをまず、約27日後のhAGOにおいて検出した後、マウス胃における生後発達の最初の数週間18と類似の34日後までに増加した7。34日後に、hFGOは、予期した通り、hAGOと同様に、MUC5AC+表面粘膜細胞及びMUC6+粘膜頸細胞の両方を含有していた(図3のa~b及び図11のa)。hFGOは、種々の内分泌細胞タイプも形成した(図3のc)が、ホルモンGASTの発現は、hAGOに特異的であったのに対し、GHRLは、hFGOにおいて10倍高く(図3のd)、正常胃内分泌パターンと一致していた19。hGOの部位特異的コンピテンスを機能的に定義するために、発明者は、誘導系を用いて、プロエンドクリン(proendocrine)転写因子NEUROG3を過剰発現させた。hGOの両サブタイプにおけるNEUROG3の発現は結果的に、パンエンドクリン(pan-endocrine)マーカーSYPならびに共通の胃ホルモンSST及びGHRLの頑強な発現を生じた(図11のc)。しかしながら、hAGOのみがGAST発現G細胞を生じるようコンピテントであり、hFGOはそうではなく(図3のe及び図11のc)、ヒト胃におけるG細胞の前庭特異的分布と一致していた19。
ベースラインで、hFGOは、プロトンポンプ(ATP4Aサブユニット及びATP4Bサブユニットからなる)を介して胃管腔を酸性化する胃底部腺に関して定義する細胞タイプである少数の傍細胞(PC)しか含有しなかった(図5のa~b)。PC集団を増大させる効率的な方法の識別は、PC集団の発生を駆動するシグナル伝達機序の理解がないので、理解しづらいままであった。発明者はそれゆえ、PC分化を調節するうえでの役割についての候補シグナル伝達経路を機能的にスクリーニングするためのプラットフォームとしてPSC由来のhFGOを用いた。スクリーニングのため、発明者は、30日後のhFGOをシグナル伝達アゴニストまたはアンタゴニストへ2日間曝露し、34日後にPC分化を分析した。シグナル伝達操作の大部分が明らかな効果を有していなかったが、PD0325901(PD03)を用いたMEK経路の一過性阻害は結果的に、ATP4A及びATB4Bの両方の実質的な上方調節を生じた(図13のa)。さらに、BMP4だけではPC遺伝子発現に影響しなかったが、PD03の効果を亢進することができた(データ非表示)。したがって、PD03/BMP4の2日間パルスは、PCマーカーATP4A、ATP4B及びGIFの迅速かつ頑強な発現を誘導するのに十分であった(図5のa~b及び図13のd)。興味深いことに、この効果は、培地からEGFまたはFGFを単純に除去することによって観察されることはなく(図13のb)、PC分化をhFGO培養へ制限することに起因するMEK/ERKの上流の内因性シグナル伝達相互作用があるようであることを示唆した。さらに、PD03/BMP4処理は、PC系譜に影響しただけであり(図13のe)、hAGOにおいてPCを誘導することはできず(図13のc)、胃上皮の初期パターン形成がその最終的な分化能を規定することをさらに強調した。
マウス実験
以下の遺伝的マウス系統をジャクソン研究室から得、シンシナティこども病院研究財団動物施設に収容し、IACUCプロトコル(0b09074)によって維持した。アキシン2:LacZ(ストック番号009120)、Shh:Cre(ストック番号005622)、及びloxPが導入されたβカテニン(ストック番号004152)。膣栓が観察された午前を0.5日胚と記し、基準交尾を用いて種々の期における胚を作製し、これをホールマウント染色または組織解剖のいずれかのために収集した。少なくとも2匹の同腹胚を、検討した各発生期において分析した。雌雄両方の胚を分析した。
ヒト胚性幹細胞株WA01(HI、WiCellから入手)をシンシナティこども病院医学センターの多能性幹細胞施設によって供給した。細胞の識別を短いタンデム反復分析(マイクロサテライトSTR分析、Applied Biosystems)によって確認し、細胞をマイコプラズマ混入について所定の通り検査した(マイコアラートマイコプラズマ検出キット、Lonza)。多能性細胞をmTesR1培地(Stem Cell Technologies)中でHESC定性化マトリゲル(BD Biosciences)上でフィーダーのない条件下で維持した。コロニーを、ディスパーゼ(Invitrogen)を用いて4日間ごとに継代した。
胃オルガノイドの定向性分化のためのプロトコルを、本発明者らの先行プロトコル7から応用し、表1は、各期についての培地及び成長因子の完全なリストを含んでいる。分化のために、hPSCを単一の細胞へと、Accutase(Stem Cell Technologies)を用いて解離し、Y-27632含有mTesR1(10μM、Stemgent)中で、ウェルあたりおよそ200,000個の細胞密度で24穴プレート中へ播種した。翌日、RPMI1640培地(Invitrogen)中のアクチビンA(100ng/ml、Cell Guidance Systems)を3日間添加することによって、細胞を胚体内胚葉(DE)へと分化させた。培地にNEAA(1×、Gibco)及び規定FBS(dFBS、Invitrogen)も1日後、2日後、及び3日後にそれぞれ0%、0.2%、及び2.0%補充した。さらに、BMP4(50ng/ml、R&D Systems)を初日に添加した。その後、細胞をCHIR99021(2μΜ、Stemgent)、FGF4(500ng/ml、R&D Systems)、及びノギン(200ng/ml、R&D systems)へ、NEAA及び2.0%dFBSを補充したRPMI1640中で3日間曝露することによって、DEを後部前腸内胚葉へと分化させた。レチノイン酸(2μΜ、Sigma Aldrich)を最終日に添加した。培地を毎日交換した。この過程は結果的に、3次元後部前腸スフェロイドの自律的な形成を生じた。
後部前腸スフェロイドを収集し、既に説明した通り36、3次元培養システムへと移した。簡潔には、スフェロイドを50μlのマトリゲル(BD Biosciences)中に懸濁し、24穴プレート中へ液滴として播種した。マトリゲルを組織培養インキュベータ中で10分間凝固させておいた後、成長因子及び/または小分子アゴニストを含有する塩基成長培地(BGM)で層状化した。BGMは、N2(1×、Invitrogen)、B27(1×、Invitrogen)、HEPES(10μM、Gibco)、L-グルタミン、ペニシリン/ストレプトマイシン、及びEGF(100ng/ml、R&D Systems)を補充した高度DMEM/F12培地(Gibco)からなった。6~9日後の間、スフェロイドをRA及びノギンとともに培養し、前庭系譜を特化させた。胃底部特化のために、CHIRをこの期の間に添加した。前庭hGOをその後、EGFのみを有するBGM中で培養した。胃底部hGOを6~30日後からCHIRへ連続的に曝露した。さらに、CHIRによって駆動される腺形態形成を亢進することが示された(データ非表示)ので、FGF10(50ng/ml、R&D Systems)を20~30日後から胃底部hGOへ添加した。20日後、オルガノイドを収集し、1:10~1:20の希釈で再播種した。
既に説明した通り、全RNAを、Nucleospin RNA IIキット(Machery Nagel)を用いて単離し、cDNAへと転換した7。Quantstudio 6(Applied Biosystems)上で、Quantitect SYBR Greenマスターミックス(Qiagen)を用いて定量的PCRを実施し、プライマー配列を以下に列挙する。
定量的PCRに使用したプライマーは以下であった。
hATP4A、順方向5'-TGGTAGTAGCCAAAGCAGCC-3'、逆方向5'-TGCCATCCAGGCTAGTGAG-3'、
hATP4B、順方向5'-ACCACGTAGAAGGCCACGTA-3'、逆方向5'-TGGAGGAGTTCCAGCGTTAC-3'、
hAXIN2、順方向5'-CTGGTGCAAAGACATAGCCA-3'、逆方向5'-AGTGTGAGGTCCACGGAAAC-3'、
hCCK、順方向5'-CGGTCACTTATCCTGTGGCT-3'、逆方向5'-CTGCGAAGATCAATCCAGCA-3'、
hCDX2、順方向5'-CTGGAGCTGGAGAAGGAGTTTC-3'、逆方向5'-ATTTTAACCTGCCTCTCAGAGAGC-3'、
hCHGA、順方向5'-TGACCTCAACGATGCATTTC-3'、逆方向5'-CTGTCCTGGCTCTTCTGCTC-3'、
hGAPDH、順方向5'-CCCATCACCATCTTCCAGGAG-3'、逆方向5'-CTTCTCCATGGTGGTGAAGACG-3'、
hGAST、順方向5'-CAGAGCCAGTGCAAAGATCA-3'、逆方向5'-AGAGACCTGAGAGGCACCAG-3'、
hGATA4、順方向5'-TCCAAACCAGAAAACGGAAGC-3'、逆方向5'-GCCCGTAGTGAGATGACAGG-3'、
hGHRL、順方向5'-GCTGGTACTGAACCCCTGAC-3'、逆方向5'-GATGGAGGTCAAGCAGAAGG-3'、
hGIF、順方向5'-CATTTTCCGCGATATTGTTG-3'、逆方向5'-GCACAGCGCAAAAATCCTAT-3'、
MRX2、順方向5'-GTGGTGTGCGCGTCGTA-3'、逆方向5'-GGCGTTCAGCCCCTACC-3'、
MRX3、順方向5'-GGAGAGAGCCGATAAGACCA-3'、逆方向5'-AGTGCCTTGGAAGTGGAGAA-3'、
MRX5、順方向5'-GGTGTGTGGTCGTAGGGAGA-3'、逆方向5'-GCTACAACTCGCACCTCCA-3'、
hMIST1、順方向5'-TGCTGGACATGGTCAGGAT-3'、逆方向5'-CGGACAAGAAGCTCTCCAAG-3'、
hMUC2、順方向5'-TGTAGGCATCGCTCTTCTCA-3'、逆方向5'-GACACCATCTACCTCACCCG-3'、
hMUC5AC、順方向5'-CCAAGGAGAACCTCCCATAT-3'、逆方向5'-CCAAGCGTCATTCCTGAG-3'、
hMUC6、順方向5'-CAGCAGGAGGAGATCACGTTCAAG-3'、逆方向5'-GTGGGTGTTTTCCTGTCTGTCATC-3'、
hPdx1、順方向5'-CGTCCGCTTGTTCTCCTC-3'、逆方向5'-CCTTTCCCATGGATGAAGTC-3'、
hSCT、順方向5'-GGTTCTGAAACCATAGGCCC-3'、逆方向5'-GTCAGGGTCCAACATGCC-3'、
hSOX2、順方向5'-GCTTAGCCTCGTCGATGAAC-3'、逆方向5'-AACCCCAAGATGCACAACTC-3'、
mCdx2、順方向5'-TCTGTGTACACCACCCGGTA-3'、逆方向5'-GAAACCTGTGCGAGTGGATG-3'、
mGata4、順方向5'-CCATCTCGCCTCCAGAGT-3'、逆方向5'-CTGGAAGACACCCCAATCTC-3'、
mGapdh、順方向5'-TTGATGGCAACAATCTCCAC-3'、逆方向5'-CGTCCCGTAGACAAAATGGT-3'、
mIrx1、順方向5'-AATAAGCAGGCGTTGTGTGG-3'、逆方向5'-CTCAGCCTCTTCTCGCAGAT-3'、
mIrx2、順方向5'-AGCTGGTATGGATAGGCCG-3'、逆方向5'-GGCTTCCCGTCCTACGTG-3'、
mIrx3、順方向5'-ATAAGACCAGAGCAGCGTCC-3'、逆方向5'-GTGCCTTGGAAGTGGAGAAA-3'、
mIrx5、順方向5'-GGAGTGTGGTCGTAGGGAGA-3'、逆方向5'-GCTACAACTCGCACCTCCA-3'、
mPdx1、順方向5'-ACGGGTCCTCTTGTTTTCCT-3'、逆方向5'-TGGATGAAATCCACCAAAGC-3'、
mPitx1、順方向5'-GTCCATGGAGGTGGGGAC-3'、逆方向5'-GCTTAGGCGCCACTCTCTT-3'、
mSox2、順方向5'-AAAGCGTTAATTTGGATGGG-3'、逆方向5'-ACAAGAGAATTGGGAGGGGT-3'、
mTrp63、順方向5'-AGCTTCTTCAGTTCGGTGGA-3'、逆方向5'-CCTCCAACACAGATTACCCG-3'。
組織を4%パラホルムアルデヒド中、4℃で一晩固定した後、PBSで完全に洗浄した。ホールマウント免疫蛍光染色のため、胚を既に説明した通り加工した37。簡潔には、胚をDentのブリーチ液(4:1:1のEtOH:DMSO:30% H2O2)中で、室温で2時間透過処理し、メタノール連続洗浄を通じて再水和させた。次に、胚を1時間ブロッキングし、1次抗体中で一晩4℃でインキュベートし、PBS中で洗浄し、1次抗体中で一晩4℃でインキュベートし、完全に洗浄した。パラフィン包埋のために、組織をエタノール連続洗浄で脱水し、キシレン中で洗浄した後、パラフィン中に包埋した。染色のため、スライドを脱パラフィン化し、再水和させた。抗原賦活化をクエン酸緩衝液中で、スチーマーの中で45分間実施した。1次抗体を4℃で一晩インキュベートした。1次抗体後、スライドをPBS中で洗浄した後、2次抗体(1:500の希釈)とともに1時間室温でインキュベートした。2次抗体(Jackson ImmunoResearch Laboratories)をロバ中で作製し、Alexa Fluor488、594、または647へ共役させた。
免疫蛍光染色に使用する抗体を、抗原、宿主種、製造元及びカタログ番号、ならびに染色に使用する希釈とともに列挙する。Atp4b、ウサギ、Santa Cruz sc84304、1:500;Cdh1、ヤギ、R&D Systems AF648、1:500;Cdh1、マウス、BD Biosciences 610182、1:500;Cdx2、マウス、Biogenex MU392A、1:500、Cldn18、ウサギ、Sigma HP A018446、1:200;Ctnnb1、ウサギ、Santa Cruz sc7190、1:100;FoxF1、ヤギ、R&D Systems F4798、1:500、ガストリン、ウサギ、Dako A0568、1:1,000;Gata4、ヤギ、Santa Cruz sc1237、1:200;Gif、ウサギ、Sigma HP A040774、1:100;Ghrl、ヤギ、Santa Cruz scl0368、1:200;ヒスタミン、ウサギ、Immunostar 22939、1:1,000;Krt8、ラット、DSHBトロマール-s;1:100;Mist1、ウサギ、Sigma HP A047834、1:200;Muc5ac、マウス、Abcam ab3649、1:500;Muc6、マウス、Abcam ab49462、1:100;Pdx1、ヤギ、Abcam ab47383、1:5,000;Pgc、ヒツジ、Abcam ab31464、1:10,000;Sst、ヤギ、Santa Cruz sc7819、1:100;Syp、モルモット、シナプス系101004、1:1,000;ビメンチン、ヤギ、Santa Cruz sc7557、1:200
ニコンAIRsi倒立型共焦点顕微鏡上で、共焦点撮像を実施した。ホールマウント撮像のため、胚をメタノール中で脱水し、Murrayのクリア液(2:1の安息香酸ベンジル:ベンジルアルコール)中で透明にした直後に撮像した。染色後、スライドをFluoromount G(SouthernBiotech)でマウントし、室温で一晩空気乾燥させた。
透過型電子顕微鏡のために、hGOを既に説明した通り加工した7。簡潔には、オルガノイドを3%グルタルアルデヒド中で固定し、0.1Mカコジル酸ナトリウム緩衝液中で洗浄し、1時間の4%四酸化オスミウムについてインキュベートした。これらをその後、洗浄した後、エタノール中で連続脱水し、酸化プロピレン/LX112中で最終的に包埋した。次に、組織を切片化し、2%酢酸ウラニル、次いでクエン酸鉛で染色した。画像を日立透過電子顕微鏡上で可視化した。
製造元の説明書により、ヒトペプシノーゲンI型(PGI)ELISAキット(Thermo Scientific,EHPGI)を用いてELISAを行った。簡潔には、34日後のhGOを細胞回収溶液(Coming)中に4℃で1時間収集及びインキュベートした後、PBS中で洗浄した。オルガノイドをRIPA緩衝液で可溶化した後、高速で室温で30分間激しくボルテックスした。溶解液をペレット化し、上清を-80℃で収集及び保存した。ELISAのために、試料及び標準物質を技術的な複製物中で実施した。反応物をμQuantマイクロプレートリーダー(Bio Tek)上で測定した。450nmでの吸光度を測定し、570nmの吸光度を減算した。
酸分泌アッセイを既に説明した通り実施した(Schumacher et al,2015)。hGOをチャンバー付きのカバーガラス(Thermo Scientific)中で生育させ、チャンバーを倒立型共焦点顕微鏡(Zeiss LSM710)上に置き、実験を5%CO2及び37℃の条件下で実施した(インキュベーションチャンバー、PeCon、ドイツ国エルバッハ)。
統計的な有意性を、対形成していないスチューデントT検定、または一元配置分散分析をDunnettの多重比較事後検査とともに用いて判定した。0.05未満のp値を有意とみなした。
統計分析を使用しないで、実験試料の規模を決め、特別な無作為化法を使用せず、研究者は実験中盲検ではなかった。統計方法及び基準を図の凡例において説明する。胃底部hGOの分化のためのプロトコルを、実験室における7名の独立したユーザによって、20回超、完了を成功させた。事例全部において、示されるデータは、複数の実験を代表する単一の実験に由来する。
以下の例は、本明細書の教示が組み合わされ得または適用され得る種々の非徹底的な方法に関する。以下の例は、本出願においてまたは本出願書類のその後の提出において、いかなるときにも呈され得るいかなる請求項の網羅も制限するよう企図するものではないことは理解されるべきである。ディスクレーマーは企図していない。以下の例は、単に説明目的以下のために提供されている。本明細書の種々の教示が数多くの他の方法において配置及び適用され得ることは熟慮される。いくつかの変法が、以下の例において引用されるある特定の特徴を省略し得ることも熟慮される。それゆえ、以下に引用される態様または特徴はいずれも、本発明者らによって、または本発明者らに対して関心のある相続人によって、後日明白に示されていない限り、決定的とみなされるべきではない。何らかの請求項が、本出願において、または以下に引用されるもの以外のさらなる特徴を含む本出願と関連するその後の出願において存在する場合、当該さらなる特徴は、特許性に関するいかなる理由についても追加されたと仮定されてはならない。
b)3次元後部前腸スフェロイドを、成長因子、Wntシグナル伝達経路活性化因子、EGFシグナル伝達経路活性化因子、BMPシグナル伝達経路阻害因子、及びレチノイン酸を有する基底膜マトリックス(例えば、マトリゲル)中で、胃底部hGO(hFGO)を含む胃底部系譜を誘導するのに十分な第2の期間懸濁し、
c)ステップb)のhFGOをwnt経路活性化因子及びEGFシグナル伝達経路活性化因子の存在下で、第3の期間培養し、
d)ステップcのhFGOをwntシグナル伝達経路活性化因子、EGFシグナル伝達経路活性化因子、及びFGF10とともに第4の期間培養し、
e)ステップdのhFGOをMEK阻害因子と、機能的胃底部細胞タイプを含む胃底部組織を形成するのに十分な期間の第5の期間接触させる(MEK阻害因子は、例えば、PD0325901であり得る)。
当該hFGOは、3次元後部前腸スフェロイドを、基底膜マトリックス中で、成長因子、wnt経路活性化薬、EGFシグナル伝達経路活性化因子、BMPシグナル伝達経路阻害因子、及びレチノイン酸と、当該3次元後部前腸スフェロイドを前記hFGOへ変換するのに十分な期間接触させることによって得られ、
当該3次元後部前腸スフェロイドは、哺乳類胚体内胚葉(DE)細胞をwnt経路活性化薬、FGFシグナル伝達経路活性化薬、BMPシグナル伝達経路阻害因子、及びレチノイン酸と接触させることによって得られる。
1.Lancaster,M.A.& Knoblich,J.A.Organogenesis in a dish:modeling development and disease using organoid technologies.Science 345,1247125-1247125(2014)。
Claims (12)
- ヒト胃底部オルガノイド(hFGO)における傍細胞の分化を誘導するインビトロ方法であって、前記hFGOにおけるATP4A、ATP4B、および/またはGIF発現のアップレギュレーションを誘導するために十分な期間前記hFGOをMEK阻害因子に接触させる工程を含み、前記hFGOは、
a)哺乳類胚体内胚葉(DE)細胞を、wntシグナル伝達経路活性化因子、FGFシグナル伝達経路活性化因子、BMPシグナル伝達経路阻害因子、及びレチノイン酸と、前記胚体内胚葉から3次元後部前腸スフェロイドを形成するのに十分な期間接触させる工程と、
b)前記3次元後部前腸スフェロイドを、基底膜マトリックス中で成長因子、wntシグナル伝達経路活性化因子、EGFシグナル伝達経路活性化因子、BMPシグナル伝達経路阻害因子、及びレチノイン酸とともに、胃底部系譜を誘導し、それによって前記hFGOを製造するのに十分な期間懸濁させる工程と、
c)工程bの前記hFGOをwntシグナル伝達経路活性化因子及びEGFシグナル伝達経路活性化因子とともに11日間±24時間である期間培養する工程と、
d)工程cの前記hFGOを前記wntシグナル伝達経路活性化因子、前記EGFシグナル伝達経路活性化因子、及びFGF10とともに10日間±24時間である期間培養する工程と、
を含む方法にしたがって調整され、
前記MEK阻害因子がPD0325901(PD03)であり、
前記wntシグナル伝達経路活性化因子は、Wnt1、Wnt2、Wnt2b、Wnt3、Wnt3a、Wnt4、Wnt5a、Wnt5b、Wnt6、Wnt7a、Wnt7b、Wnt8a、Wnt8b、Wnt9a、Wnt9b、Wnt10a、Wnt10b、Wnt11、Wnt16、GSK3-ベータ阻害薬、及びCHIR99021から選択され、
前記FGFシグナル伝達経路活性化因子は、FGF2、FGF4、FGF5、及びFGF8であり、
前記BMPシグナル伝達経路阻害因子は、ノギン、ドルソモルフィン、LDN189、及びDMH-1から選択され、及び
前記EGFシグナル伝達経路活性化因子は、EGFである、方法。 - 請求項1記載の方法において、前記hFGOにおけるATP4A、ATP4B、および/またはGIF発現のアップレギュレーションを誘導するために十分な期間は2日間±24時間である、方法。
- 請求項1または2記載の方法において、前記胚体内胚葉から3次元後部前腸スフェロイドを形成するのに十分な期間は3日間±24時間であり、前記レチノイン酸は、前記胚体内胚葉から3次元後部前腸スフェロイドを形成するのに十分な期間±24時間の3日目に添加される、方法。
- 請求項1~3のいずれかに記載の方法において、胃底部系譜を誘導し、それによって前記hFGOを製造するのに十分な期間は3日間±24時間である、方法。
- 請求項1~4のいずれかに記載の方法において、hFGOsにおける傍細胞の分化の誘導が、ヒスタミンとの処理に応じて前記hFGOsの管腔のpHが低下し、ファモチジンまたはオメプラゾールで前処理した場合に前記低下が抑制されることを特徴とする、方法。
- 請求項1~5のいずれか1項に記載の方法において、前記hFGOsにおける傍細胞の分化の誘導が、前記hFGOにおける傍細胞の密集した腺の存在によって特徴づけられる、方法。
- 請求項1~6のいずれか1項に記載の方法において、前記hFGOsを前記MEK阻害因子と接触させる工程が、前記hFGOsにおける主細胞または内分泌細胞の分化に影響を与えない、方法。
- 請求項1~7のいずれか1項に記載の方法であって、さらに、前記hFGOsをBMP4シグナル伝達の活性化因子と接触させる工程を含む、方法。
- 請求項8記載の方法において、前記BMP4シグナル伝達の活性化因子がBMP4である、方法。
- 請求項1~9のいずれか1項に記載の方法において、前記hFGOが胚体内胚葉に由来する、方法。
- 請求項10記載の方法において、前記胚体内胚葉が、胚性幹細胞および人工多能性幹細胞から選択される前駆細胞に由来する、方法。
- 請求項1~11のいずれか1項に記載の方法によって誘導されるhFGO。
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