JP6781355B1 - 洞房結節様ペースメーカー心筋細胞および心室様心筋細胞を作製および使用するための方法 - Google Patents
洞房結節様ペースメーカー心筋細胞および心室様心筋細胞を作製および使用するための方法 Download PDFInfo
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Abstract
Description
本願は、参照によりその全文が本明細書に組み込まれる2015年2月17日出願の米国特許仮出願第62/117,107号の優先権に基づく米国特許法第119条の利益を主張する特許協力条約出願である。
配列表の組み込み
a.BMP成分、随意にBMP4を含み、随意にRho関連プロテインキナーゼ(ROCK)阻害剤をさらに含む胚様体培地中でhPSCを一定期間インキュベートして胚様体を生成する工程;
b.BMP成分、随意にBMP4、およびアクチビン成分、随意にアクチビンA、および随意にFGF成分、随意にbFGFを含む中胚葉誘導培地中で胚様体を一定期間インキュベートして心血管中胚葉細胞を生成する工程;
c.心血管中胚葉細胞を、
i.
1.選択された量を上回るBMP成分、随意にBMP4、およびレチノイン酸(RA)、および随意に1または複数のFGF阻害剤、WNT阻害剤、随意にIWP2、VEGFおよびアクチビン/ノーダル阻害剤、随意にSB−431542を含む心臓誘導培地中で;一定期間インキュベートして、TBX18を発現する心血管前駆細胞を生成し、該心血管中胚葉細胞が好ましくはFGF阻害剤とともにインキュベートされ、そのFGF阻害剤が心臓誘導段階の全部または一部に提供され;かつ
2.VEGFを含む基礎培地中で心血管前駆細胞を一定期間インキュベートして、SANLCMに富む心筋細胞集団を生成する工程;または
ii.
1.1または複数のWNT阻害剤、随意にIWP2、およびVEGF;ならびに随意にFGF成分および/またはアクチビン/ノーダル阻害剤、随意にSB−431542を含む心臓誘導培地中で;一定期間インキュベートして、NKX2−5を発現する心血管前駆細胞を生成し、かつ
2.VEGFを含む基礎培地中で心血管前駆細胞を一定期間インキュベートして、NKX2−5poscTNTpos細胞に富み、SANLCMを実質的に欠く心筋細胞集団を生成する工程;および
d.随意に、心筋細胞特異的表面マーカーを使用して心筋細胞集団を単離する工程、随意に、該マーカーは、シグナル調節タンパク質α(SIRPA)、および胸腺細胞分化抗原1(THY−1/CD90)であり、随意に該単離された集団はSIRPAposCD90negである。
a.BMP成分、随意にBMP4を含み、随意にRho関連プロテインキナーゼ(ROCK)阻害剤をさらに含む胚様体培地中で、hPSCを一定期間インキュベートして胚様体を生成する工程;
b.BMP成分、随意にBMP4、およびアクチビン成分、随意にアクチビンAおよび随意にFGF成分、随意にbFGFを含む中胚葉誘導培地中で、胚様体を一定期間インキュベートして心血管中胚葉細胞を生成する工程;
c.
i.心血管中胚葉細胞を、選択された量を上回るBMP成分、随意にBMP4、およびレチノイン酸(RA)、および随意に1または複数のFGF阻害剤、WNT阻害剤、随意にIWP2、VEGFおよびアクチビン/ノーダル阻害剤、随意にSB−431542を含む心臓誘導培地中で;一定期間インキュベートしてTBX18を発現する心血管前駆細胞を生成する工程;該心血管中胚葉細胞は、好ましくはFGF阻害剤とともにインキュベートされ、そのFGF阻害剤は心臓誘導段階の全部または一部に提供される;かつ
ii.VEGFを含む基礎培地中で心血管前駆細胞を一定期間インキュベートして、SANLCMに富む心筋細胞集団を生成する工程;ならびに
d.随意に、心筋細胞特異的表面マーカーを使用してSANLCMに富む心筋細胞集団を単離する工程、随意に、該マーカーは、シグナル調節タンパク質α(SIRPA)および胸腺細胞分化抗原1(THY−1/CD90)であり、随意に該単離された集団はSIRPAposCD90negである。
a.BMP成分、随意にBMP4を含み、随意にRho関連プロテインキナーゼ(ROCK)阻害剤をさらに含む胚様体培地中で、hPSCを一定期間インキュベートして胚様体を生成する工程;
b.BMP成分、随意にBMP4、およびアクチビン成分、随意にアクチビンAおよび随意にFGF成分、随意にbFGFを含む中胚葉誘導培地中で、胚様体を一定期間インキュベートして心血管中胚葉細胞を生成する工程;
c.
i.心血管中胚葉細胞を、1または複数のWNT阻害剤、随意にIWP2、およびVEGF;ならびに随意にFGF成分および/またはアクチビン/ノーダル阻害剤、随意にSB−431542を含む心臓誘導培地中で;一定期間インキュベートして、NKX2−5を発現する心血管前駆細胞を生成し;かつ
ii.VEGFを含む基礎培地中で心血管前駆細胞を一定期間インキュベートして、NKX2−5poscTNTpos細胞に富み、SANLCMを実質的に欠く心筋細胞集団を生成する工程;ならびに
d.随意に、心筋細胞特異的表面マーカーを使用して心筋細胞集団を単離する工程、随意に、該マーカーは、シグナル調節タンパク質α(SIRPA)および胸腺細胞分化抗原1(THY−1/CD90)であり、随意に該単離された集団はSIRPAposCD90negである。
用語「洞房結節様心筋細胞」または「SANLCM」とは、本明細書において、洞房結節(SAN)細胞特異的マーカーTBX18、TBX3、SHOX2およびISL1を発現し、ペースメーカー活性を有する心筋細胞のサブタイプをさす。SAN細胞のように、SANLCMは、低レベルのNKX2−5を発現し、心室様心筋細胞などのその他の心筋細胞サブタイプよりも速い拍動数(例えば、1分当たりの拍動数)を有する。
用語「FGF阻害剤」は、本明細書において、どんなFGF受容体阻害剤(FGFR1,2,3,4)またはFGFシグナル伝達阻害剤(すなわち、下流のp38 MAPK阻害剤)も意味し、それには限定されるものではないが、PD173074(Tocris)およびSU5402(Torcis)およびp38 MAPK阻害剤SB203580(Tocris)が含まれる。
SANLCMを豊富に含むかまたは実質的に含まない細胞集団を生成および単離するための方法が本明細書において開示される。この細胞型を指定するための成分および条件、ならびにこれらの細胞の出現をモニターするためのマーカーが記載される。
a.心血管中胚葉細胞を、選択された量を上回るBMP成分、随意にBMP4、およびレチノイン酸(RA)、および随意に1または複数のWNT阻害剤、随意にIWP2、VEGF;およびアクチビン/ノーダル阻害剤、随意にSB−431542を含む心臓誘導培地中で;一定期間インキュベートして、TBX18を発現する心血管前駆細胞を生成する工程;
b.VEGFを含む基礎培地中で心血管前駆細胞を一定期間インキュベートして、SANLCMに富む心筋細胞集団を生成する工程;および
c.随意に、心筋細胞特異的表面マーカーを使用してSANLCMに富む心筋細胞集団を単離する工程、随意に、該マーカーはシグナル調節タンパク質α(SIRPA)であり、随意に、該単離された集団はSIRPAposCD90negである。
a.心血管中胚葉細胞を1または複数のWNT阻害剤、随意にIWP2、およびVEGF;および随意にFGF成分および/またはアクチビン/ノーダル阻害剤、随意にSB−431542を含む心臓誘導培地中で;一定期間インキュベートして、心血管前駆細胞を生成する工程;
b.VEGFを含む基礎培地中で心血管前駆細胞を一定期間インキュベートして、NKX2−5poscTNTposに富み、SANLCMを実質的に欠く心筋細胞集団を生成する工程;および
c.随意に、心筋細胞特異的表面マーカーを使用してSANLCMを実質的に欠くNKX2−5poscTNTposである心筋細胞集団を単離する工程、随意に、該マーカーはシグナル調節タンパク質α(SIRPA)であり、随意に、該単離された集団はSIRPAposCD90negである。
a.BMP成分、随意にBMP4を含み、随意にRho関連プロテインキナーゼ(ROCK)阻害剤をさらに含む胚様体培地中で、hPSCを一定期間インキュベートして胚様体を生成する工程;
b.BMP成分、随意にBMP4、およびアクチビン成分、随意にアクチビンAおよび随意にFGF成分、随意にbFGFを含む中胚葉誘導培地中で、胚様体を一定期間インキュベートして心血管中胚葉細胞を生成する工程;
c.心血管中胚葉細胞を、選択された量を上回るBMP成分、随意にBMP4、およびレチノイン酸(RA)、および随意に1または複数のFGF阻害剤、WNT阻害剤、随意にIWP2、VEGFおよびアクチビン/ノーダル阻害剤、随意にSB−431542を含む心臓誘導培地中で;一定期間インキュベートしてTBX18を発現する心血管前駆細胞を生成する工程;該心血管中胚葉細胞は、好ましくはFGF阻害剤とともにインキュベートされ、そのFGF阻害剤は心臓誘導段階の全部または一部に提供される;
d.VEGFを含む基礎培地中で心血管前駆細胞を一定期間インキュベートして、SANLCMに富む心筋細胞集団を生成する工程;ならびに
e.随意に、心筋細胞特異的表面マーカーを使用してSANLCMに富む心筋細胞集団を単離する工程、随意に、該マーカーは、シグナル調節タンパク質α(SIRPA)であり、随意に該単離された集団はSIRPAposCD90negである。
a.SIRPAマーカー陽性心筋細胞を単離すること;および/または
b.NKX2−5発現に対して陰性かつ/またはCD90発現に対して陰性の心筋細胞を選択すること
を含む。
a.SIRPAマーカー陽性心筋細胞を単離すること;および/または
b.NKX2−5発現に対して陽性かつ/またはCD90発現に対して陰性の心筋細胞を選択すること
を含む。
もう一つの態様には、a)hPSCからNKX2−5レポーター構築物を含む心筋細胞集団を、随意に本明細書に記載される方法に従って作製すること、およびb)NKX2−5陰性または陽性心筋細胞を選択することを含む、SANLCMまたはVLCMを心筋細胞集団から単離する方法が含まれる。
a.本明細書に記載される方法に従ってSANLCMを生成する工程;
b.SANLCMと候補試験薬物を接触させる工程;
c.SANLCMの拍動数、活動電位特性および/またはイオン電流を測定する工程;
d.SANLCMの拍動数、活動電位特性および/またはイオン電流を、候補試験薬物で処置していない対照SANLCMと比較する工程;および
e.候補薬物として対照細胞と比較した拍動数および/または活動電位を調節する候補試験薬物を選択する工程
を含む、候補薬物を特定する方法である。
方法
hPSCの維持および分化
ヒト多能性幹細胞株(Hes3 NKX2−5:GFP)10H7(NIH登録番号006132により承認されたHESC株)およびMSC−iPS111を、記載されるように培養した12。心臓系列への分化のため、確立されたプロトコール6を、次の変更を行って用いた(図1A)。80%コンフルエントのhPSC培養物を、単一細胞に分離させ、1ng/ml BMP4および10μM ROCK阻害剤を含有するStemPro−34培地に懸濁し、18時間オービタルシェーカーでインキュベートして、胚様体(EB)を生成した。翌日(分化の1日目)、EBを中胚葉誘導培地:10ng/ml BMP4、6ng/ml アクチビンA、5ng/ml bFGFを含有するStemPro−34に移した。分化の3日目に、EBを、IMDMを用いて1回洗浄し、心臓誘導培地:0.5μM IWP2、10ng/ml VEGF、および随意に5.4μM SB−431542(SB、アクチビン/ノーダル/TGFβ阻害剤)を含有するStemPro−34に懸濁した。分化の5日目に、EBを、基礎培地:5ng/ml VEGFを含有するStemProに切り替えた。培地は、分化の12日目までVEGFを添加し、4日毎に変えた。別途示されない場合、全てのサイトカインはR&D systemsより購入した。EBは、12日目まで5%CO2、5%O2、90%N2の低酸素条件下で、そして培養期間の残りを5%CO2の、通常の空気条件下で培養した。
EBを、中胚葉段階(分化3日目)に、各細胞への効率的なサイトカイン供給を確保するようにTrypLE(Life Technologies)を合理的に使用して単一細胞に分離させた。再集合してEBとなるように、細胞を示されたサイトカインおよび5μM ROCK阻害剤とともに、96ウェル低クラスタープレートに80,000細胞/ウェルで入れた(図2A)。分化の5日目にEBを24ウェル低クラスタープレートに移し、上記のように基礎培地中で培養し続けた。
3日目〜12日目にEBを分離するために、TrypLEを使用した。12〜30日目に、TypLE分離の前にEBを1mg/ml 2型コラーゲン(Worthington)を含有するHANK緩衝液中で穏やかに振盪しながら室温で一晩インキュベートした。細胞を、抗PDGFRa−PE(1:20)抗KDR−APC(1:10)、(R&D Systems)、抗SIRPA−PeCy(クローンSE5A5、1:1000、Biolegend)、抗CD90−APC(BD)、cTnTの抗心臓アイソフォーム(クローン13−11;1:2000、Thermo Fischer)、ウサギ抗ヒトNKX2−5(1:800、Cell Signaling Technology)、ヤギ抗マウスIgG APC(1:250、BD)、ヤギ抗マウスIgG PE(1:200、Jackson ImmunoResearch)、ロバ抗ウサギIgG A647(Life Technologies)で5×106細胞/mlの濃度で染色した。細胞表面マーカーには、5%FCSを含有するPBS中で染色を行った。細胞内染色には、細胞を、4%PFAを用いて4℃で10分間固定し、5%FCSおよび0.5%サポニン(cTNT)または0.3%TritonX(NKX2−5)を含有するPBS中で染色した。LSR II フローサイトメーター(BD)を用いて染色した細胞を分析した。細胞選別には、細胞を、5%FCSを含有するIMDM中で保持し、MoFlo(BD)およびInflux(BD)ソーターを用いて106細胞/mlの濃度で選別した。その後のRNA単離のためにPFA固定細胞を選別するには、染色緩衝液および細胞収集のためのPBSに5mM DTTおよび100U/ml RNaseOUT(Life Technologies)を添加した。全ての緩衝液成分は、RNAseフリーであって、試料は厳密に氷上で保持され、PFA固定から3時間以内に選別された。データは、FlowJoソフトウェア(Tree Star)を用いて分析した。
細胞は、4%PFAで10分間4℃で固定し、0.3%Triton X、200mMグリセリンを使用して室温で20分間透過処理した。試料を、ブロッキング緩衝液(BB):10%ロバ血清、2%BSA、0.1%Triton X中で室温で30分間ブロッキングした。以下の一次抗体を、BB中で一晩4℃でインキュベートした:cTnTの抗心臓アイソフォーム(クローン13−11;1:100、Thermo Fischer)、ウサギ抗ヒトMLC2v(1:100)、ウサギ抗ヒトSHOX2(1:200)、(Abcam)、ウサギ抗ヒトTBX3(1:100)、ヤギ抗ヒトTbx18(1:50)、(Santa Cruz)。それぞれの二次抗体を、30分間室温でインキュベートした:ロバ抗マウスIgG A647(1:400)、ロバ抗ウサギIgG A467(1:400)、(Life Technologies)、ヤギ抗マウスIgG Cy3(1:400)、ロバ抗ウサギIgG Cy3(1:800)、ロバ抗ヤギIgG Cy3(1:800)、(Jackson ImmunoResearch)。DAPIを使用して細胞核を対比染色し、Fluorescent Mounting Medium(Dako)を用いてスライドを取り付けた。Olympus FluoView 1000レーザー走査型共焦点顕微鏡およびFV10−ASWソフトウェアをイメージングに使用した。
全RNAは、RNAqueous−micro Kit(Ambion)とそれに続くDNase消化ステップ(Ambion)を用いて単離した。PFAで固定した細胞からのRNAの単離には、RecoverAll Kit(Ambion)を使用した。500ng〜1μgのRNAを、ランダムヘキサマーおよびOligo(dT)プライマーとSuperscript III Reverse Transcriptase(Life Technologies)を用いてcDNAに逆転写した。QPCRsは、QuntiFast SYBR Green PCR Kit(Qiagen)を製造業者の使用説明書に従って使用して、EP RealPlex MasterCycler(Eppendorf)で実施した。25ng/μlから2.5pg/μlに及ぶヒトゲノムDNA標準物質の10倍希釈系列を使用してPCRの効率を評価し、以前に記載されたようにハウスキーピング遺伝子TBPと比較して各遺伝子のコピー数を計算した13。プライマー配列は表3に記載される。ヒト胎児心臓組織妊娠段階17をNovogenix Laboratoriesより購入した。心室、洞房結節および房室結節組織を切開した後、RNAをTrizol法(Life Technologies)を用いて単離し、DNアーゼ(Ambion)で処置した。
活動電位および膜電流は、Axopatch増幅器を使用して、基準電流および電圧クランプ技術によって測定された。データ獲得および分析には、pCLAMPソフトウェア(Molecular Devices)を使用した。ホウケイ酸ガラス微小電極は、ピペット溶液で満たされると2〜5MΩの先端抵抗を有した。直列抵抗および細胞キャパシタンスは70%まで補償された。自発的活動電位、ファニー電流(If)および内向き整流性カリウム電流電流(IKr)は、穿孔パッチ法(ナイスタチン)14を次の浴溶液:NaCl 124、KCl 5.4、CaCl2 1.2、MgCl2 1、およびグルコース 5、Hepes 10(pH7.4、NaOHで調節)(単位mM)とともに使用して37℃で記録された。ピペット溶液は、NaCl 5、KCl 10、グルコン酸カリウム 130、MgCl2 1、およびHepes 10(pH7.4、KOHで調節)(単位mM)から成っていた。ナトリウム電流(INa)は、より良好な電圧制御のために電流振幅を低下させるために、低Na+を含有するカルシウムフリーの浴溶液中で全細胞破裂パッチ法を使用して25℃で記録された:NaCl 30、TEA−Cl 115、CaCl2 0.01、MgCl2 1、およびグルコース 5、Hepes 10(pH7.4、NaOHで調節)(単位mM)。ピペット溶液は:Cs−アスパラギン酸塩 112、CsCl 20、MgCl2 1、Mg−ATP 5、Cs−EGTA 10、Hepes 10(pH7.4、CsOHで調節)(単位mM)から成った。個々の電流を誘発するための電圧プロトコールは、それぞれの図に示される。
試験管内ペーシング実験のために、1×106VLCMをマトリゲルでコーティングされた多電極アレイ(MEA)に播種し、5〜7日間培養して、電気的に統合された単層を形成した。テトラメチルローダミンメチルエステル(TMRM)で標識したSANLCMまたはVLCM(対照)集合体(3×104細胞)を、これらの確立したVLCM単層上の特定の場所に置いた。電気シグナル(電場電位)を、Multichannel systems増幅器、加熱装置およびCardio−2Dソフトウェアを用いて37℃の基礎培地中で記録した。電気シグナル伝播のグレースケールマップを、Cardio−2D+ソフトウェア(Multichannelsystems)を使用して生成した。
データは、平均値±標準誤差として提示した。統計分析は、スチューデントT検定を用いて実施した。結果は、p<0.05(*/#)で有意であるとみなされ、p<0.01(**/##)で非常に有意であるとみなされた。
hESC由来の心血管細胞を生成するために、T(BRACHYURY)を発現する原条様集団の形成(2〜3日目)とその後のMESP1およびPDGFRα/KDRの発現を特徴とする心臓中胚葉の誘導(3〜4日目)を含む、発達的に段階分けしたプロトコール6、15を使用した。このPDGFRα+KDR+中胚葉は、60〜90%のcTnT+心筋細胞を生じさせる(20日目)(図1Aおよび図5A、B)。このプロトコールによって、TBX18、SHOX2およびTBX3を含む洞房結節(SAN)の発生に関与する転写因子の発現16が分化の3〜8日の間にアップレギュレートされ、ペースメーカー細胞がこれらの条件下で特定化されていることが示唆される(図1B)。マウスおよびヒトの両方の研究は、SAN細胞が全心筋細胞転写因子NKX2−5を発現しない前駆細胞に由来することを示すので17−19、NKX2−5の発現に基づいてhPSC由来のペースメーカー細胞をその他の心筋細胞から識別することが可能であると仮定された。これを試験するために、HES3 NKX2−5:GFPレポーター株10を全心筋細胞表面マーカーSIRPAと組み合わせて使用して、NKX2−5+/NKX2−5−心筋細胞の存在について培養物をモニターした13。最初のNKX2−5:GFP+SIRPA+細胞は、分化の6日以内に生成され、集団のサイズは増加して16日目までに培養の60±3%となった(図1C)。また、明確なNKX2−5:GFP−SIRPA+集団が16日目までに検出された。これらの細胞の心筋細胞である性質は、cTnT染色によって確認し、それにより大きい(63±8%)NKX2−5:GFP+cTnT+集団および小さい(6±2%)NKX2−5:GFP−cTNT+集団が明らかになった。
生体内移植および生体外同時ECG記録および光学マッピングのための方法
SANLCMおよびVLCMの生体内ペースメーカー能力の評価のために、8匹の成体Fischer−344(200〜300g)ラットを使用した。動物に麻酔し(ケタミン87mg/kg、キシラジン13mg/kg)、挿管し、機械的人工呼吸を行った(O2 100%、体積1ml/kg)。左開胸術の後、1〜2×106 SANLCMまたはVLCM(低クラスター96ウェルプレートに80,000細胞/ウェルで集合)を、28Gニードルを使用して心尖部に近い左心室前壁に注入した。ヒト細胞移植片の免疫拒絶を防ぐために、動物をシクロスポリンA(13mg/kg/日)およびメチルプレドニゾロン(2mg/kg/日)で処置した。
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Claims (22)
- 心筋細胞集団を産生する方法であって、
a.心血管中胚葉細胞を、WNT阻害剤、VEGFまたはその両方を含む心臓誘導培地中で、一定期間インキュベートして、NKX2−5を発現する心血管前駆細胞を生成し;
b.VEGFを含む基礎培地中で前記心血管前駆細胞を一定期間インキュベートして、NKX2−5poscTnTpos細胞に富む心筋細胞集団を生成する工程と含む、方法。 - 前記心血管中胚葉細胞が、多能性幹細胞(PSC)から生成される、請求項1に記載の方法であって、
a.前記PSCを、BMP成分を含む胚様体培地中で一定期間インキュベートして、胚様体を生成する工程と;
b.前記胚様体を、BMP成分およびアクチビン成分を含む中胚葉誘導培地中で一定期間インキュベートして、心血管中胚葉細胞を生成する工程
を含む方法に従って、心血管中胚葉細胞が生成される、方法。 - 前記PSCがヒトPSC(hPSC)である、請求項2に記載の方法。
- 前記hPSCが、ヒト誘導多能性幹細胞(iPSC)またはヒト胚幹細胞(hESC)である、請求項3に記載の方法。
- 前記hPSCが、NKX2−5レポーター構築物を含むhPSCである、請求項3又は4に記載の方法。
- 更に、前記心筋細胞集団から心室様心筋細胞(VLCM)集団を単離する工程であって、NKX2−5レポーター構築物の発現に従って、NKX2−5陽性心筋細胞を選択する工程を含む、請求項5に記載の方法。
- 前記NKX2−5レポーター構築物が、蛍光NKX2−5レポーター構築物である、請求項5又は6に記載の方法。
- 前記心臓誘導培地は、WNT阻害剤を含む、請求項1〜7の何れか1項に記載の方法。
- 前記心臓誘導培地は、更にVEGFを含む、請求項8に記載の方法。
- 前記WNT阻害剤は、IWP2である、請求項8又は9に記載の方法。
- 前記心臓誘導培地は、更にFGF成分およびアクチビン/ノーダル阻害剤の少なくとも1種を含む、請求項1〜10の何れか1項に記載の方法。
- 前記心臓誘導培地は、更にSB−431542を含む、請求項1〜10の何れか1項に記載の方法。
- 更に、心筋細胞特異的表面マーカーを使用して前記心筋細胞集団を単離する工程を含む、請求項1〜12の何れか1項に記載の方法。
- 前記心筋細胞集団を単離する工程は、シグナル調節タンパク質α(SIRPA)および胸腺細胞分化抗原1(THY−1/CD90)を使用して行う、請求項13に記載の方法。
- 前記胚様体培地中のBMP成分が、BMP4であり、前記中胚葉誘導培地中のBMP成分が、BMP4である、請求項2に記載の方法。
- 前記胚様体培地は、更にRho関連プロテインキナーゼ(ROCK)阻害剤を含み、前記中胚葉誘導培地中のアクチビン成分は、アクチビンAであり、前記中胚葉誘導培地は、更にbFGFを含む、請求項15に記載の方法。
- 前記心血管中胚葉細胞が、多能性幹細胞(PSC)から生成される、請求項12に記載の方法であって、
a.前記PSCを、BMP成分を含む胚様体培地中で一定期間インキュベートして、胚様体を生成する工程と;
b.前記胚様体を、BMP成分およびアクチビン成分を含む中胚葉誘導培地中で一定期間インキュベートして、心血管中胚葉細胞を生成する工程と
を含む方法に従って、心血管中胚葉細胞が生成される、方法。 - 前記胚様体培地中のBMP成分が、BMP4であり、前記中胚葉誘導培地中のBMP成分が、BMP4である、請求項17に記載の方法。
- 前記胚様体培地は、更にRho関連プロテインキナーゼ(ROCK)阻害剤を含み、前記中胚葉誘導培地中のアクチビン成分は、アクチビンAであり、前記中胚葉誘導培地は、更にbFGFを含む、請求項18に記載の方法。
- 前記PSCがヒトPSC(hPSC)である、請求項19に記載の方法。
- 前記hPSCが、ヒト誘導多能性幹細胞(iPSC)またはヒト胚幹細胞(hESC)である、請求項20に記載の方法。
- 前記蛍光NKX2−5レポーター構築物が、NKX2−5:GFPレポーター構築物である、請求項7に記載の方法。
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