JPWO2014192312A1 - IPS cell-derived myocardial model cells having normal inward potassium current characteristics - Google Patents
IPS cell-derived myocardial model cells having normal inward potassium current characteristics Download PDFInfo
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Abstract
本発明の課題は、薬剤研究開発におけるQT間隔延長作用及び催不整脈性を検出する評価系に用いる、評価細胞とその樹立方法、及びそれを用いた被検物質のスクリーニング方法を提供することにある。人工多能性幹細胞(iPS細胞)に由来し、心臓トロポニンT(TnT)、コネキシン43(Cx43)、又はα−アクチニン(α−actinin)のうち少なくとも1つの細胞内因性遺伝子を発現し、かつ、導入されたKCNJ2遺伝子によりKir2.1チャネルを発現し、電気刺激を与えたときの最大拡張期電位が−85〜−65mVであり、自発的な周期的収縮活性を有さないことを特徴とする細胞を作製する。An object of the present invention is to provide an evaluation cell and its establishment method, and a screening method for a test substance using the evaluation cell, which are used in an evaluation system for detecting QT interval prolonging action and arrhythmogenicity in drug research and development. . Derived from induced pluripotent stem cells (iPS cells), expressing at least one cellular endogenous gene of cardiac troponin T (TnT), connexin 43 (Cx43), or α-actinin, and The Kir2.1 channel is expressed by the introduced KCNJ2 gene, and the maximum diastolic potential when applied with electrical stimulation is −85 to −65 mV, and has no spontaneous periodic contractile activity Make cells.
Description
本発明は、正常な内向きのカリウム電流特性を有するiPS細胞由来の心筋モデル細胞、その作製方法、及びそれを用いた心筋細胞に対して毒性作用や変調作用を有する物質のスクリーニング方法に関する。 The present invention relates to an iPS cell-derived myocardial model cell having normal inward potassium current characteristics, a method for producing the same, and a method for screening a substance having a toxic action or a modulating action on cardiomyocytes using the iPS cell-derived myocardial model cell.
動悸、失神及び不整脈として現れる、医薬品副作用のトルサード・ド・ポアント(TdP)は、心電図QT間隔延長を伴う心室性頻脈であり、致死的な予後不良の不整脈症状として知られる[非特許文献1]。TdPは、抗不整脈薬をはじめ抗腫瘍剤、抗生物質、抗精神病薬など、あらゆる医薬品投与時に起こり得る重篤な副作用であるため、創薬の過程において、QT間隔延長作用及び催不整脈性をもつ医薬品候補物質を、厳密に選別する必要がある。 Drug side effect Torsade de Pointe (TdP), which manifests as palpitations, syncope and arrhythmia, is a ventricular tachycardia accompanied by an extended electrocardiogram QT interval, and is known as an arrhythmic symptom with a fatal poor prognosis [Non-patent document 1 ]. TdP is a serious side effect that can occur during the administration of any drug, including antiarrhythmic drugs, antitumor agents, antibiotics, antipsychotics, etc., and thus has a QT interval prolonging action and arrhythmogenicity in the process of drug discovery. It is necessary to strictly select drug candidates.
TdPは、QT間隔、つまり心室の興奮から再分極までの時間の延長を伴う。QT間隔延長は、心筋細胞膜にあるイオンチャネルの障害による、イオン電流の変化と密接に関係しており、中でもカリウムイオン(K+)チャネル異常の関与が大きいと考えられている。心筋細胞に存在するK+チャネルには大きく分けて2種類あり、ひとつは電位依存性K+チャネル(Kv)と呼ばれ、心臓活動電位の第1相に関与する一過性外向き電流と、第3相に関与する遅延整流K+電流(IK)を制御する[図1]。もうひとつは内向き整流K+チャネル(Kir:inwardly rectifying K channel)と呼ばれ[図1]、IK1を含む3種類の内向き整流K+電流を制御する。IK1電流のチャネルはKir2.1とKir2.2のヘテロ4量体で構成され、静止膜電位の維持、及び活動電位最終局面での静止膜電位への再分極化といった機能を司る[図1]。TdP is accompanied by a QT interval, ie, an extension of the time from ventricular excitement to repolarization. QT interval prolongation is closely related to changes in ionic current due to damage to ion channels in the myocardial cell membrane, and it is considered that potassium ion (K + ) channel abnormalities are particularly involved. There are roughly two types of K + channels present in cardiomyocytes, one of which is called a voltage-dependent K + channel (Kv), a transient outward current involved in the first phase of the cardiac action potential, Control the delayed rectifier K + current (IK) involved in the third phase [FIG. 1]. The other is called an inwardly rectifying K + channel (Kir: Inwardly rectifying K channel) [FIG. 1] and controls three types of inward rectifying K + currents including IK1. The channel of the IK1 current is composed of a heterotetramer of Kir2.1 and Kir2.2 and manages functions such as maintenance of resting membrane potential and repolarization to resting membrane potential in the final phase of action potential [Fig. 1]. .
QT間隔延長を誘発する薬物の作用機序としては、心筋細胞における、薬物による遅延整流K+電流(IK)の速い成分(IKr)の抑制が知られている。IKrを形成するK+チャネルのサブユニット分子であるhERG(HumanEther-a-go-go Related Gene)で構成されるhERGチャネルは、薬物との結合や細胞外のカリウムイオンレベルの低下に対して敏感であり、どちらの場合も結果としてQT間隔延長を引き起こす。IKrの抑制、すなわち、hERGチャネルの阻害が、QT間隔延長を引き起こすという知見により、日米欧各国の医療行政当局は、日米欧医薬品規制調和国際会議(ICH)による、前臨床開発での心疾患安全性確立に対する勧告(ICH S7B:心室再分極遅延/QT間隔延長の薬学的臨床前評価ガイドライン)に基づき、新薬開発においてhERGチャネルに対する安全性試験の実施を義務付けている[非特許文献2]。As a mechanism of action of a drug that induces QT interval prolongation, suppression of a fast component (IKr) of delayed rectifier K + current (IK) by a drug in cardiomyocytes is known. HERG channel composed of hERG (HumanEther-a-go-go Related Gene), a subunit molecule of K + channel that forms IKr, is sensitive to drug binding and decreased extracellular potassium ion level. In both cases, this results in QT interval extension. With the knowledge that suppression of IKr, ie, inhibition of hERG channel, causes QT interval prolongation, the medical administration authorities in Japan, the US and Europe are thinking about preclinical development by the International Conference on Harmonization of Pharmaceutical Regulations (ICH). Based on a recommendation for establishing disease safety (ICH S7B: preclinical guideline for evaluation of ventricular repolarization delay / QT interval prolongation), it is obliged to conduct safety tests on hERG channels in the development of new drugs [Non-patent Document 2] .
hERGチャネルに対する安全性試験に用いるモデル細胞の選択には、細胞のもつ電気生理学的特性に注意を要するが、入手可能性などの理由から、ヒト以外の生物種の心筋細胞、もしくは、ヒトイオンチャネル遺伝子を心筋細胞以外のヒト細胞株へ異所性に発現させた細胞が、従来用いられてきた。これら従来型モデル細胞は、前者ではヒトとは異なるイオンチャネルの分布が問題であり、また後者の場合は、心筋細胞と異なる環境下による、細胞内Ca2+ハンドリングの関与や、心筋細胞内に豊富なミトコンドリアの影響を無視した条件である点などが、ヒト心筋のモデル細胞として扱う際に問題となる[非特許文献3]。In selecting a model cell for use in a safety test for a hERG channel, attention must be paid to the electrophysiological characteristics of the cell. However, for reasons such as availability, cardiomyocytes of biological species other than humans or human ion channels Cells in which genes are ectopically expressed in human cell lines other than cardiomyocytes have been used in the past. In these former model cells, the distribution of ion channels different from that of humans is a problem in the former, and in the latter case, the involvement of intracellular Ca 2+ handling under different environments from cardiomyocytes, The point of ignoring the influence of abundant mitochondria is a problem when handled as a model cell of human myocardium [Non-patent Document 3].
全ての組織に分化し得るヒト人工多能性幹細胞(hiPS細胞)から分化誘導された心筋細胞は、ヒトの心臓から単離した心筋細胞に近い電気生理学的性質を有すると考えられ、新たなヒト心筋モデル細胞として脚光を浴びている。hiPS細胞の創薬応用により、正確なin vitroスクリーニングが可能となれば、開発の後期段階でQT間隔延長作用の発覚により対象から外される多くの医薬品候補物質を、早期の段階でふるい分けることが可能となり、大幅な開発費用削減が見込まれる。 Cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS cells) capable of differentiating into all tissues are considered to have electrophysiological properties close to those of cardiomyocytes isolated from the human heart. It is in the limelight as a myocardial model cell. If accurate in vitro screening becomes possible through the application of drug discovery for hiPS cells, many drug candidates that are excluded from the target due to the detection of QT interval prolongation will be screened at an early stage. As a result, significant development cost reduction is expected.
しかしながら、hiPS由来心筋細胞の特性を精査すると、以下のような性質を示すことから、ヒト心筋モデル細胞としての使用には理想的とは言い難い。すなわち、hiPS細胞から胚様体(EB:Embryoid body)を形成させ[図2]、得られたhiPS由来心筋細胞には、結節型、心房筋型、心室筋型の活動電位特性を示す細胞が混在し[図3]、活動電位の幅に大きなばらつきが認められ[図4A]、内向き整流K+電流(IK1)が微弱であり[図4C]、静止膜電位にほとんど影響せず[図4B]、臨床濃度のhERGチャネル阻害薬E4031により自律拍動が停止する[図5]ため、スクリーニングに用いた場合、正確な判定に支障をきたす恐れがある。However, scrutinizing the characteristics of hiPS-derived cardiomyocytes shows the following properties and is not ideal for use as human myocardial model cells. That is, an embryoid body (EB) is formed from hiPS cells [FIG. 2], and the obtained hiPS-derived cardiomyocytes include cells having action potential characteristics of nodule type, atrial muscle type, and ventricular muscle type. Mixed [FIG. 3], large variation in action potential width was observed [FIG. 4A], inward rectification K + current (IK1) was weak [FIG. 4C], and hardly affected the resting membrane potential [FIG. 4B] Autonomic pulsation is stopped by the clinical concentration of hERG channel inhibitor E4031 [FIG. 5], and therefore, when used for screening, accurate determination may be hindered.
また、hiPS由来心筋細胞は、自律拍動性が特徴のひとつであるが[図3,4]、哺乳類発生の過程における胎生初期の心筋細胞では、一つひとつの細胞が自律的に活動電位を形成するものの、胎生後期においては自律性が消滅することから、成人ヒト心筋細胞は、刺激伝導系から電気刺激を受け取り活動電位を形成する、受動的な細胞であるはずと考えられる。従って、hiPS細胞由来心筋細胞は、[図3,4]に示すような自律的な活動電位を発している点で、非常に幼若な、胎生初期の心筋細胞に近い性質を持つことを意味し、この様な細胞を成人の心筋モデル細胞として使用するには適さない。 In addition, hiPS-derived cardiomyocytes are one of the features of autonomic pulsatility [Figs. 3 and 4], but in the early embryonic cardiomyocytes in the process of mammalian development, each cell autonomously forms an action potential. However, since autonomy disappears in the late embryonic period, adult human cardiomyocytes are thought to be passive cells that receive electrical stimulation from the stimulation conduction system and form action potentials. Therefore, hiPS cell-derived cardiomyocytes have a very young, early fetal cardiomyocyte nature in that they generate autonomous action potentials as shown in [Figs. 3 and 4]. However, such cells are not suitable for use as adult myocardial model cells.
QT間隔延長作用を有する、hERGチャネル阻害薬のE4031を用いて、心室筋型hiPS由来心筋細胞の、活動電位に対するE4031作用を測定すると、低濃度(10,30nM)の曝露の場合は、[図5 A]に示すような自動的活動電位の発生頻度の低下を呈するが、E4031濃度を徐々に高めながら曝露濃度を100nMまで上げると、静止膜電位が浅くなり、すなわち、静止膜電位値が0に近づき、hiPS由来心筋細胞は、脱分極の不能により活動電位の停止に陥る[図5 B]。E4031洗浄により活動電位が復活するので、これは測定ダメージによる活動電位発生力の消失ではなく、100nMのE4031で活動電位が停止したことを示す[図5 B]。生体への使用では、同濃度のE4031投与により心停止を引き起こさないことが、臨床的に確認されていることから、in vitro電気生理試験における、hiPS由来心筋細胞に対するE4031の作用濃度が、ヒト生体内心筋細胞に対する実際の作用濃度と異なることを意味している。また、E4031が本来持つ、QT間隔延長作用の検出も困難である[図5 B]。 Using the hERG channel inhibitor E4031 having a QT interval prolongation effect, the E4031 effect on the action potential of ventricular myPS cells was measured, and in the case of low concentration (10, 30 nM) exposure, 5 A], the occurrence frequency of the automatic action potential is decreased. However, when the exposure concentration is increased to 100 nM while gradually increasing the E4031 concentration, the resting membrane potential becomes shallow, that is, the resting membrane potential value is 0. The hiPS-derived cardiomyocytes fall into the action potential cessation due to the inability to depolarize [FIG. 5B]. Since the action potential is restored by E4031 washing, this indicates that the action potential was stopped at 100 nM E4031 rather than the disappearance of the action potential generation force due to the measured damage [FIG. 5B]. Since it has been clinically confirmed that administration of E4031 at the same concentration does not cause cardiac arrest when used in vivo, the action concentration of E4031 on hiPS-derived cardiomyocytes in in vitro electrophysiological tests is It means that it is different from the actual concentration of action on in vivo cardiomyocytes. In addition, it is difficult to detect the QT interval extending action inherent to E4031 [FIG. 5B].
従って、成人の心筋細胞とhiPS由来心筋細胞との間では、i)成人の心室心筋細胞では、洞房結節(ペースメーカー)からの刺激無しでは拍動しないのに対して、hiPS由来心筋細胞は自律拍動する点、ii)最大拡張期電位(MDP)が成人の心室心筋細胞では約−80mVであるのに対して、hiPS由来心筋細胞では−40〜−50mVである点、iii)hERG/K+チャネルブロックにより、成人の心室心筋細胞は活動電位の再分極相が延長、すなわち心室筋活動電位持続時間(APD:QT間隔)が延長するのに対して、hiPS由来心筋細胞では、脱分極の不能により活動電位の停止を引き起こす点、において大きく異なっている[図6]。Therefore, between adult cardiomyocytes and hiPS-derived cardiomyocytes, i) adult ventricular cardiomyocytes do not beat without stimulation from the sinoatrial node (pacemaker), whereas hiPS-derived cardiomyocytes Ii) The maximum diastolic potential (MDP) is about −80 mV in adult ventricular cardiomyocytes, whereas it is −40 to −50 mV in hiPS-derived cardiomyocytes, iii) hERG / K + Channel blocking allows adult ventricular cardiomyocytes to prolong the action potential repolarization phase, ie, ventricular muscle action potential duration (APD: QT interval), whereas hiPS-derived cardiomyocytes cannot depolarize. Is significantly different in that the action potential stops due to [FIG. 6].
既報では、心毒性スクリーニング用モデル細胞として、hERGチャネルを遺伝子導入した例が挙げられるが[特許文献1,2]、hERG遺伝子を発現させる対象が、非ヒト哺乳動物由来のチャイニーズハムスター卵巣(CHO)細胞である点で、上述した従来のモデル細胞に共通する問題点を有する。また、ヒト胚幹細胞由来機能性心筋細胞も、心毒性スクリーニング用モデル細胞として発表されているが[特許文献3]、イオンチャネルに関する遺伝子改変はなく、物理的に収縮している細胞を対象としている点で、本発明とは異なる。 In the previous report, as examples of model cells for cardiotoxicity screening, there are examples in which hERG channels have been introduced [Patent Documents 1 and 2], but the subject to express the hERG gene is a Chinese hamster ovary (CHO) derived from a non-human mammal. It has a problem common to the above-described conventional model cell in that it is a cell. In addition, human embryonic stem cell-derived functional cardiomyocytes have also been published as model cells for cardiotoxicity screening [Patent Document 3], but are not genetically modified with respect to ion channels and are intended for physically contracting cells. This is different from the present invention.
また、受託試験サービスとして商業的に提供されている「ヒトiPS由来心筋による心筋毒性試験」(QTempo;登録商標,株式会社リプロセル)[非特許文献4]では、i)ヒトiPS由来心筋細胞塊(胚様体)そのものを試験に使用している、ii)心筋細胞が自律拍動しており幼若である、iii)試験に使用されるヒトiPS由来心筋細胞は、結節、心房、心室の全ての型が混在していることから、本発明の細胞と異なり、心毒性を予想する上で、本発明に比べ外挿性に劣ると予想される。 In addition, in “a myocardial toxicity test using human iPS-derived myocardium” (Q Tempo; registered trademark, Reprocell Co., Ltd.) [Non-Patent Document 4] commercially provided as a contracted test service, i) human iPS-derived cardiomyocyte mass ( The embryoid body) is used for the test itself, ii) the cardiomyocytes are autonomously pulsating and young, iii) the human iPS-derived cardiomyocytes used for the test are all nodules, atria and ventricles Therefore, unlike the cells of the present invention, it is expected to be inferior in extrapolation compared to the present invention in predicting cardiotoxicity.
最新の報告によると、胚性幹細胞(ES細胞)由来心筋細胞は、Kir2.1チャネル遺伝子の導入により、強いBa2+感受性IK1電流が発生し、自律拍動性を失い静止状態に変化することが示されている[非特許文献5]。しかしながら、ヒトiPS由来心筋細胞とヒトES由来心筋細胞では、50%再分極時の活動電位持続時間(APD50)に、約1.37倍の開きがあることから(iPS由来:382+/-38ms,n=36、ES由来:278+/-28ms,n=64)[Lopez-Redondo F, Kurokawa J, et al., Human ES- and iPS-derived cardiomyocytes. A comparative electrophysiological study. 57th Biophysical Society Annual Meeting, Philadelphia, Biophys J, 104, 298a. (Feb 3-6, 2013)]、両者における電気生理学的性質の差が示唆されており、かつ、iPS由来心筋細胞において、Kir2.1チャネルを強発現させる系が検討されたという報告は未だ例がない。According to the latest report, embryonic stem cell (ES cell) -derived cardiomyocytes can develop a strong Ba 2+ sensitive IK1 current due to the introduction of Kir2.1 channel gene, lose their autonomous pulsatility and change to a quiescent state. [Non-Patent Document 5]. However, in human iPS-derived cardiomyocytes and human ES-derived cardiomyocytes, the action potential duration (APD50) at the time of 50% repolarization has an opening of about 1.37 times (iPS origin: 382 +/− 38 ms). , N = 36, ES origin: 278 +/− 28 ms, n = 64) [Lopez-Redondo F, Kurokawa J, et al., Human ES- and iPS-derived cardiomyocytes. A comparative electrophysiological study. 57th Biophysical Society Annual Meeting , Philadelphia, Biophys J, 104, 298a. (Feb 3-6, 2013)], suggesting a difference in electrophysiological properties between the two and strongly expressing Kir2.1 channel in iPS-derived cardiomyocytes There are no reports of the system being considered.
臨床で経験される、hERGチャネル阻害による心電図QT延長とは、すなわち心室心筋の活動電位持続時間(APD)が延長することに他ならない。従来技術には、hERGチャネル阻害によるQT延長作用の検出に用いるモデル細胞として、単純にhERGチャネルを発現させた非ヒト由来細胞株の作出例のみ存在した[特許文献1,2]。 The electrocardiogram QT prolongation by hERG channel inhibition experienced in clinical practice is nothing but the prolongation of action potential duration (APD) of ventricular myocardium. In the prior art, as a model cell used for detection of the QT prolonging effect by hERG channel inhibition, only a non-human cell line in which hERG channel is expressed was simply created [Patent Documents 1 and 2].
本発明の課題は、薬剤研究開発におけるQT間隔延長作用及び催不整脈性を検出する評価系に用いる評価細胞とその樹立方法、及びそれを用いた被検物質のスクリーニング方法を提供することにある。 An object of the present invention is to provide an evaluation cell used in an evaluation system for detecting QT interval prolonging action and arrhythmogenicity in drug research and development, a method for establishing the cell, and a screening method for a test substance using the cell.
本発明者らは、上記課題を解決すべく鋭意研究を行う過程において、内向き整流K+電流(IK1)に対するK+チャネルKir2.1をコードする遺伝子の、KCNJ2に着目した。アデノウイルスを用いてhiPS由来心筋細胞にKir2.1チャネルを発現させることで、強い内向き整流K+電流(IK1)が発生し、hiPS由来心筋細胞がもつ自律拍動の特性が失われ、電気刺激依存的に活動電位が発生するという知見を得た。またエピソーマル型ベクターを用いて遺伝子導入することもできる。また、最大拡張期電位(MDP)が、KCNJ2遺伝子発現により約−50mVから−70mVへシフトし、成熟したヒト心室筋型心筋細胞の性質に近い、電気生理学的特性を示すことを見出した。更に、hiPS由来心筋細胞では、hERG阻害によりMDPが浅くなり短縮することがあった活動電位持続時間が、KCNJ2発現細胞ではhERG阻害による活動電位持続時間の延長を呈し、すなわち、hERG阻害によるQT間隔延長のin vitroでの再現に成功した。本発明はこれらの知見に基づき完成するに至ったものである。In the course of conducting intensive research to solve the above problems, the present inventors focused on KCNJ2, a gene encoding the K + channel Kir2.1 for the inward rectifier K + current (IK1). By expressing Kir2.1 channel in hiPS-derived cardiomyocytes using adenovirus, strong inward rectification K + current (IK1) is generated, and the characteristics of the autonomous pulsation possessed by hiPS-derived cardiomyocytes are lost. We have found that action potentials are generated in a stimulus-dependent manner. It is also possible to introduce genes using episomal vectors. In addition, it was found that the maximum diastolic potential (MDP) was shifted from about −50 mV to −70 mV by KCNJ2 gene expression, and exhibited electrophysiological characteristics close to those of mature human ventricular myocardial cells. Furthermore, in hiPS-derived cardiomyocytes, the action potential duration that MDP became shallow and shortened due to hERG inhibition showed an increase in action potential duration due to hERG inhibition in KCNJ2-expressing cells, that is, the QT interval due to hERG inhibition. The extension was successfully reproduced in vitro. The present invention has been completed based on these findings.
すなわち、本発明は、
〔1〕人工多能性幹細胞(iPS細胞)に由来し、心臓トロポニンT(TnT)、コネキシン43(Cx43)、又はα−アクチニン(α−actinin)のうち少なくとも1つの細胞内因性遺伝子を発現し、かつ、導入されたKCNJ2遺伝子によりKir2.1チャネルを発現する心筋モデル細胞に関する。That is, the present invention
[1] It is derived from an induced pluripotent stem cell (iPS cell) and expresses at least one cell endogenous gene of cardiac troponin T (TnT), connexin 43 (Cx43), or α-actinin (α-actinin). And a myocardial model cell expressing a Kir2.1 channel by the introduced KCNJ2 gene.
また本発明は、
〔2〕自発的な周期的収縮活性を有さないことを特徴とする上記〔1〕に記載の心筋モデル細胞、
〔3〕生理的細胞内外液を使用した条件において、電気刺激を与えたときの最大拡張期電位が、−85〜−65mVであることを特徴とする上記〔1〕又は〔2〕に記載の心筋モデル細胞、
〔4〕Kir2.1チャネルの発現が、ウイルスベクターに導入されたKCNJ2遺伝子の発現であることを特徴とする上記〔1〕〜〔3〕のいずれかに記載の心筋モデル細胞、
〔5〕人工多能性幹細胞(iPS細胞)がヒト由来であることを特徴とする上記〔1〕〜〔4〕のいずれかに記載の心筋モデル細胞に関する。The present invention also provides
[2] The myocardial model cell according to [1] above, which has no spontaneous periodic contractile activity,
[3] The condition according to [1] or [2] above, wherein the maximum diastolic potential when an electrical stimulus is applied is -85 to -65 mV in a condition using a physiological intracellular / extracellular fluid. Myocardial model cells,
[4] The myocardial model cell according to any one of [1] to [3] above, wherein the expression of the Kir2.1 channel is expression of a KCNJ2 gene introduced into a viral vector,
[5] The myocardial model cell according to any one of [1] to [4] above, wherein the induced pluripotent stem cell (iPS cell) is derived from a human.
さらに本発明は、
〔6〕以下の(a)〜(e)の工程を備えたことを特徴とする心筋モデル細胞の作製方法であって、
1)人工多能性幹細胞(iPS細胞)から心筋細胞又は心筋前駆細胞に分化させる工程(a);
2)工程(a)により得られた心筋細胞又は心筋前駆細胞を含む胚様体若しくはコロニーを、単一細胞に分離する工程(b);
3)KCNJ2遺伝子を組み込み、Kir2.1チャネルを発現可能なベクターを調製する工程(c);
4)工程(b)により分離した細胞を、分離直後から1時間以内に、工程(c)で調製したウイルスベクターに感染させる工程(d);
5)心臓トロポニンT(TnT)、コネキシン43(Cx43)、又はα−アクチニン(α−actinin)のうち少なくとも1つの細胞内因性遺伝子を発現し、かつ、Kir2.1チャネルを発現する細胞を選択する工程(e)を含む方法、
〔7〕ベクターがアデノウイルスベクターであることを特徴とする上記〔6〕に記載の方法、
〔8〕ベクターがエピソーマル型ベクターであることを特徴とする上記〔6〕に記載の方法、
〔9〕以下の(A)〜(C)の工程を備えたことを特徴とする心筋細胞に対して毒性作用及び/又は変調作用を有する物質のスクリーニング方法であって、
1)上記〔1〕〜〔5〕のいずれかに記載の心筋モデル細胞と被検物質とを接触させる工程(A);
2)電気生理学的試験手法を用いて、被検物質に起因する心筋細胞に対する毒性作用及び/又は変調作用を検出する工程(B);
3)工程(B)の検出結果に基づき、被検物質の心筋細胞に対する毒性作用及び/又は変調作用の有無を判定する工程(C);を含む方法、
〔10〕工程(B)における毒性作用及び/又は変調作用が、hERG電流阻害活性であることを特徴とする上記〔9〕に記載の方法に関する。Furthermore, the present invention provides
[6] A method for producing a myocardial model cell comprising the following steps (a) to (e):
1) a step of differentiation from induced pluripotent stem cells (iPS cells) into cardiomyocytes or myocardial progenitor cells (a);
2) A step (b) of separating the embryoid body or colony containing cardiomyocytes or myocardial progenitor cells obtained in step (a) into single cells;
3) A step of preparing a vector capable of expressing the Kir2.1 channel by incorporating the KCNJ2 gene (c);
4) A step (d) of infecting the cells separated in step (b) with the virus vector prepared in step (c) within 1 hour immediately after the separation;
5) Select a cell that expresses at least one cellular endogenous gene of cardiac troponin T (TnT), connexin 43 (Cx43), or α-actinin and expresses the Kir2.1 channel A method comprising step (e),
[7] The method according to [6] above, wherein the vector is an adenovirus vector,
[8] The method according to [6] above, wherein the vector is an episomal vector,
[9] A screening method for a substance having a toxic action and / or a modulating action on cardiomyocytes characterized by comprising the following steps (A) to (C):
1) A step (A) of contacting the myocardial model cell according to any one of [1] to [5] above with a test substance;
2) A step (B) of detecting a toxic effect and / or a modulation effect on cardiomyocytes caused by a test substance using an electrophysiological test method;
3) a step (C) of determining the presence or absence of a toxic effect and / or a modulation effect on the cardiomyocytes of the test substance based on the detection result of the step (B),
[10] The method according to [9] above, wherein the toxic action and / or modulation action in the step (B) is hERG current inhibitory activity.
本発明により、hiPS由来心筋細胞の問題点であった、自律拍動性すなわち未成熟さが解消され、成人ヒト心室心筋レベルに最大拡張期電位が深く、かつ、成人ヒト心室心筋細胞と同様のhERG阻害薬に対するQT間隔延長作用を示す、心筋モデル細胞が提供される。すなわち、心毒性のある薬剤の選別、及び、臨床試験に用いる用量の設定といった、創薬に必須のin vitroスクリーニングにおいて、本発明のモデル細胞を用いることで、外挿性の高いスクリーニング結果が期待できる。 According to the present invention, autopulsation, i.e., immaturity, which has been a problem of hiPS-derived cardiomyocytes, is resolved, the maximum diastolic potential is deep at the level of adult human ventricular myocardium, and A myocardial model cell is provided that exhibits a QT interval prolonging effect on a hERG inhibitor. That is, screening results with high extrapolation are expected by using the model cells of the present invention in in vitro screening essential for drug discovery, such as selection of drugs with cardiotoxicity and setting of doses for clinical trials. it can.
本発明は、薬剤研究開発における被検物質の心筋細胞への影響を試験する際に有利に使用することができる心筋モデル細胞やその作製法に関し、本発明の心筋モデル細胞は、最大拡張期電位の深さ、hERGチャネル阻害による活動電位持続時間の延長傾向、及び自律拍動しない点において、成人心筋細胞が持つ電気生理学的特性と共通し、創薬スクリーニングに用いた場合、結果として得られる情報の質の向上が期待できる。 The present invention relates to a myocardial model cell that can be advantageously used when testing the influence of a test substance on cardiomyocytes in drug research and development, and a method for producing the same. The myocardial model cell of the present invention has a maximum diastolic potential. Information obtained when used for drug discovery screening, in common with the electrophysiological characteristics of adult cardiomyocytes in terms of the depth of action, the tendency of the action potential duration to be prolonged due to hERG channel inhibition, and the point of no autonomous pulsation Improvement of quality can be expected.
本発明の心筋モデル細胞としては、人工多能性幹細胞(iPS細胞)に由来し、心臓トロポニンT(TnT)、コネキシン43(Cx43)、又はα−アクチニン(α−actinin)のうち少なくとも1つの細胞内因性遺伝子を発現し、かつ、導入されたKCNJ2遺伝子によりKir2.1チャネルを発現する細胞であれば特に制限されないが、自発的な周期的収縮活性を有さない細胞や、生理的細胞内外液を使用した条件において、電気刺激を与えたときの最大拡張期電位が、−85〜−65mVである細胞や、Kir2.1チャネルの発現が、ウイルスベクターに導入されたKCNJ2遺伝子の発現である細胞や、人工多能性幹細胞(iPS細胞)がヒト由来である細胞が好ましい。 The myocardial model cell of the present invention is derived from an induced pluripotent stem cell (iPS cell), and at least one cell of cardiac troponin T (TnT), connexin 43 (Cx43), or α-actinin (α-actinin) The cell is not particularly limited as long as it is a cell that expresses an endogenous gene and expresses the Kir2.1 channel by the introduced KCNJ2 gene. However, cells that do not have spontaneous periodic contractile activity, and physiological intracellular and extracellular fluids Cells having a maximum diastolic potential of -85 to -65 mV when electrical stimulation is applied, or cells in which the expression of the Kir2.1 channel is the expression of the KCNJ2 gene introduced into a viral vector Alternatively, cells in which the induced pluripotent stem cells (iPS cells) are derived from humans are preferable.
また、本発明の心筋モデル細胞の作製方法としては、iPS細胞から心筋細胞又は心筋前駆細胞に分化させる工程(a);工程(a)により得られた心筋細胞又は心筋前駆細胞を含む胚様体若しくはコロニーを、単一細胞に分離する工程(b);KCNJ2遺伝子を組み込み、Kir2.1チャネルを発現可能なウイルスベクターを調製する工程(c);工程(b)により分離した細胞を、分離直後から1時間以内に、工程(c)で調製したウイルスベクターに感染させる工程(d);心臓トロポニンT(TnT)、コネキシン43(Cx43)、又はα−アクチニン(α−actinin)のうち少なくとも1つの細胞内因性遺伝子を発現し、かつ、Kir2.1チャネルを発現する細胞を選択する工程(e)の各工程を備えた方法であれば特に制限されないが、上記ウイルスベクターがアデノウイルスベクターであることが好ましい。かかる本発明の作製方法により、上記本発明の心筋モデル細胞を得ることができる。 The method for producing a myocardial model cell of the present invention includes the step (a) of differentiating iPS cells into cardiomyocytes or myocardial progenitor cells; an embryoid body containing cardiomyocytes or myocardial progenitor cells obtained by step (a) Alternatively, the step of separating colonies into single cells (b); the step of preparing a viral vector that can incorporate the KCNJ2 gene and express the Kir2.1 channel (c); Within 1 hour of infection with the viral vector prepared in step (c) (d); at least one of cardiac troponin T (TnT), connexin 43 (Cx43), or α-actinin If it is a method comprising each step of the step (e) of selecting a cell expressing a cell endogenous gene and expressing a Kir2.1 channel Although not particularly limited, the virus vector is preferably an adenovirus vector. The myocardial model cell of the present invention can be obtained by the production method of the present invention.
本発明の心筋モデル細胞は、内因性遺伝子から心筋細胞及び心筋前駆細胞の特異的マーカーである心臓トロポニンT(TnT)、コネキシン43(Cx43)、及びα−アクチニン(α−actinin)の内、少なくとも1つ、より好ましくは2つ、特に好ましくは3つ以上の細胞内因性遺伝子を発現するが、これらに加えて心臓トロポニンI(cTnI)、筋節ミオシン重鎖(MHC)、ミオシン軽鎖(MLC)、N−カドヘリン、β1−アドレナリン受容体(β1−AR)、心房性ナトリウム利尿ペプチド(ANF)、ミオグロビン、クレアチンキナーゼMB(CK−MB)、GATA−4(心筋転写因子)、NKX2.5(心筋転写因子)、及びMEF−2ファミリー(心筋転写因子)から選ばれる1又は2種以上の特異的マーカーを発現する細胞が好ましい。 The myocardial model cell of the present invention comprises at least one of cardiac troponin T (TnT), connexin 43 (Cx43), and α-actinin, which are specific markers of cardiac muscle cells and cardiac muscle progenitor cells from endogenous genes. Express one, more preferably two, particularly preferably three or more cellular endogenous genes, but in addition to these, cardiac troponin I (cTnI), sarcomeric myosin heavy chain (MHC), myosin light chain (MLC) ), N-cadherin, β1-adrenergic receptor (β1-AR), atrial natriuretic peptide (ANF), myoglobin, creatine kinase MB (CK-MB), GATA-4 (myocardial transcription factor), NKX2.5 ( Myocardial transcription factor), and one or more specific markers selected from the MEF-2 family (myocardial transcription factor) Current to cells are preferred.
本発明の心筋モデル細胞は、形態学的に明瞭な心筋細胞の特徴を有する。すなわち、免疫染色によって検出可能な、筋節構造に特有の横紋を有し[図13 B]、紡錘形、円形、三角形、多角形などの形態を示し得る。 The myocardial model cell of the present invention has morphologically distinct cardiomyocyte characteristics. That is, it has a striation specific to the sarcomere structure that can be detected by immunostaining [FIG. 13B], and can show forms such as a spindle shape, a circle shape, a triangle shape, and a polygon shape.
上記iPS細胞としては、山中ら[Cell. 2007 Nov 30;131(5):861-72.]によって記載される方法で作製されたヒト人工多能性幹細胞の他、京都大学から提供されているhiPS細胞株の201B7、253G1、並びに、Cellular Dynamics International,Inc.(CDI)社から販売されている分化心筋細胞iCellの様に、樹立された細胞系統の形態でも提供される細胞を例示することができる。かかるiPS細胞として、初代コロニーから直接得られる細胞や、もしくは分化のためにすぐに使用可能な他の方法で作製されたiPS細胞も用いることができる。また、iPS細胞の由来としては、ヒトの他、アカゲザル、カニクイザルなどの非ヒト霊長類、ラット、マウス、ウサギ、ヒツジ、ブタ、ウシ、ウマ、ヤギ、ネコ、イヌなどの実験動物、家畜、ペットを好適に挙げることができる。 The iPS cells are provided by Kyoto University in addition to human induced pluripotent stem cells prepared by the method described by Yamanaka et al. [Cell. 2007 Nov 30; 131 (5): 861-72.]. Examples include cells provided in the form of established cell lines, such as the hiPS cell line 201B7, 253G1, and differentiated cardiomyocytes iCell sold by Cellular Dynamics International, Inc. (CDI). it can. As such iPS cells, cells directly obtained from primary colonies or iPS cells prepared by other methods that can be used immediately for differentiation can be used. In addition to humans, iPS cells can be derived from humans, non-human primates such as rhesus monkeys and cynomolgus monkeys, laboratory animals such as rats, mice, rabbits, sheep, pigs, cows, horses, goats, cats and dogs, livestock, and pets. Can be preferably mentioned.
本発明におけるKir2.1チャネルとしては、KCNJ2遺伝子にコードされた、心筋細胞における内向き整流K+チャネルの構成タンパク質の一つであって、配列番号1(UniProtKB Accession No.:P63252)で表わされるアミノ酸配列からなるタンパク質、又はこれら配列と95%以上の同一性を有する同効物を好適に挙げることができる。ここで同効物とは、細胞に発現させた場合、配列番号1で表わされるアミノ酸配列を有するタンパク質と実質的に同質のKir2.1活性を有するポリペプチドいい、上記Kir2.1活性とは、カリウムイオンチャネルとして機能し、内向き整流性K+イオン電流を発生させる活性を意味する。また、上記実質的に同質の活性とは、その活性が性質的に、例えば電気生理学的あるいは薬理学的に、同質であることを示す。The Kir2.1 channel in the present invention is one of the constituent proteins of the inward rectifier K + channel encoded by the KCNJ2 gene in cardiomyocytes and represented by SEQ ID NO: 1 (UniProtKB Accession No .: P63252) Preferred examples include proteins comprising amino acid sequences, or synergistic substances having 95% or more identity with these sequences. Here, the synergistic product refers to a polypeptide having Kir2.1 activity that is substantially the same quality as the protein having the amino acid sequence represented by SEQ ID NO: 1 when expressed in a cell. It means an activity that functions as a potassium ion channel and generates an inward rectifying K + ion current. The substantially homogeneous activity means that the activity is qualitatively, for example, electrophysiologically or pharmacologically.
かかるKir2.1チャネルのアミノ酸配列情報は、UniProt(http://www.uniprot.org/)等のデータベース検索により、適宜入手することができ、IRK2_HUMAN(UniProtKB Accession No.:P63252)として登録されたものを、具体的に例示することができる。また、Kir2.1チャネルの塩基配列情報は、NCBI(http://www.ncbi.nlm.nih.gov/guide/)等のデータベース検索により、適宜入手することができ、GenBank Accession No.:NM_000891.2 GI:22095339として登録されたものを、具体的に例示することができる。 The amino acid sequence information of the Kir2.1 channel can be obtained as appropriate by searching a database such as UniProt (http://www.uniprot.org/) and registered as IRK2_HUMAN (UniProtKB Accession No .: P63252). Things can be specifically exemplified. Further, the base sequence information of Kir2.1 channel can be appropriately obtained by database search such as NCBI (http://www.ncbi.nlm.nih.gov/guide/), and GenBank Accession No .: NM_000891 .2 What is registered as GI: 22095339 can be specifically exemplified.
本発明においてKCNJ2遺伝子とは、Kir2.1チャネルをコードする塩基配列からなるポリヌクレオチドをいう。本発明のKCNJ2遺伝子は、配列番号2(GenBank Accession No.:NM_000891.2 GI:22095339)で表わされる塩基配列からなるポリヌクレオチド、又はこれら配列と95%以上の同一性を有する同効物を好適に挙げることができる。ここで同効物とは、配列番号2で表わされる塩基配列を有するポリヌクレオチドと実質的に同質のKir2.1活性を有するポリペプチドをコードする塩基配列からなるポリヌクレオチドをいう。 In the present invention, the KCNJ2 gene refers to a polynucleotide having a base sequence encoding a Kir2.1 channel. The KCNJ2 gene of the present invention is preferably a polynucleotide comprising the base sequence represented by SEQ ID NO: 2 (GenBank Accession No .: NM — 000891.2 GI: 22095339), or an equivalent product having 95% or more identity with these sequences. Can be listed. Here, the synergistic substance refers to a polynucleotide comprising a base sequence encoding a polypeptide having Kir2.1 activity substantially the same as the polynucleotide having the base sequence represented by SEQ ID NO: 2.
本発明の心筋モデル細胞の作製方法において用いられる、KCNJ2遺伝子を含むウイルスベクターとしては、上記KCNJ2遺伝子を宿主細胞内に保持し、該KCNJ2遺伝子にコードされるKir2.1チャネルを発現することができるウイルスベクターであれば特に制限されず、かかるウイルスベクターとしては、モロニーマウス白血病ウイルスなどをベースにしたレトロウイルス、ヒト免疫不全1型ウイルス(HIV−1)などをベースにしたレンチウイルス、アデノウイルス、アデノ随伴ウイルス(AAV)などを由来とするウイルスベクターが挙げられ、特に、サイトメガロウイルスプロモーターを含有するpAd/CMV/V5−DESTアデノウイルスベクターなどを好適に挙げることができる。 As a viral vector containing the KCNJ2 gene used in the method for producing a myocardial model cell of the present invention, the KCNJ2 gene can be retained in a host cell and the Kir2.1 channel encoded by the KCNJ2 gene can be expressed. The viral vector is not particularly limited as long as it is a viral vector. Examples of such viral vectors include retroviruses based on Moloney murine leukemia virus, lentiviruses based on human immunodeficiency type 1 virus (HIV-1), adenoviruses, A viral vector derived from an adeno-associated virus (AAV) or the like can be mentioned, and a pAd / CMV / V5-DEST adenoviral vector containing a cytomegalovirus promoter can be particularly preferably mentioned.
例えば、アデノウイルスベクターにKCNJ2遺伝子を導入すると、導入遺伝子を好ましくは1ヶ月以上、より好ましくは2ヶ月以上、持続的かつ長期的にiPS由来心筋細胞上で発現させることができる。一般的に、アデノウイルスベクターは、導入遺伝子が染色体外でエピゾーマルに存在し自律複製することはないため、一過性の遺伝子発現を示し、よって細胞が分裂するごとに導入遺伝子の発現は弱まる。しかし、ヒト心筋細胞は、生後約1カ月で分裂能を喪失するという性質のため、アデノウイルスベクターによる導入遺伝子の長期発現が期待でき、本発明において遺伝子導入されたiPS由来心筋細胞においても、約2ヶ月のKCNJ2遺伝子発現を確認している。より長期的なKCNJ2遺伝子の発現には、非分裂細胞への感染力があり、かつ、導入遺伝子の一部で染色体への組み込みを期待できる、アデノ随伴ウイルスベクター(AAV)を有利に使用することができる。 For example, when the KCNJ2 gene is introduced into an adenovirus vector, the transgene can be expressed on iPS-derived cardiomyocytes continuously and long-term, preferably for 1 month or more, more preferably for 2 months or more. In general, an adenovirus vector exhibits transient gene expression because the transgene exists episomally outside the chromosome and does not replicate autonomously, and thus the expression of the transgene is weakened each time the cell divides. However, human cardiomyocytes can be expected to exhibit long-term expression of a transgene by an adenovirus vector because of the property of losing division ability at about one month after birth. In iPS-derived cardiomyocytes transfected with the present invention, about Two months of KCNJ2 gene expression has been confirmed. For long-term expression of KCNJ2 gene, adeno-associated virus vector (AAV) that has the ability to infect non-dividing cells and that can be expected to be integrated into the chromosome of a part of the transgene is advantageously used. Can do.
KCNJ2遺伝子導入アデノウイルスベクターは、ヒト胎児由来腎臓細胞(HEK細胞)の様な非心筋細胞に対しても、Ba2+感受性の内向き整流K+電流を発生させることができ[図19]、HEK細胞の他に、チャイニーズハムスター卵巣(CHO)細胞、アフリカミドリザル腎臓由来COS−7細胞、アフリカツメガエル卵母細胞などもBa2+感受性の内向き整流K+電流を発生させることができる。The KCNJ2 gene-introduced adenoviral vector can generate Ba 2+ sensitive inward rectifier K + currents even for non-cardiomyocytes such as human embryonic kidney cells (HEK cells) [FIG. 19], HEK In addition to cells, Chinese hamster ovary (CHO) cells, African green monkey kidney-derived COS-7 cells, Xenopus oocytes, and the like can also generate Ba 2+ sensitive inward rectifier K + currents.
上記ウイルスベクターは、選択可能なマーカーを含んでもよく、選択可能なマーカーとして、具体的にはアンピシリン、ネオマイシン、カナマイシン、ハイグロマイシン、ピューロマイシン、クロラムフェニコールなどの薬剤に対する耐性遺伝子のほか、GFP、EGFP(Enhanced Green Fluorescent Protein)などの蛍光タンパク質などを例示することができる。 The viral vector may contain a selectable marker. Specific examples of the selectable marker include genes for resistance to drugs such as ampicillin, neomycin, kanamycin, hygromycin, puromycin, chloramphenicol, and GFP. And fluorescent proteins such as EGFP (Enhanced Green Fluorescent Protein).
KCNJ2遺伝子導入アデノウイルスベクターの構築に用いることができる、組換えクローニング技術としては、TAクローニングや制限酵素を用いるクローニングのほかに、大腸菌におけるラムダファージの部位特異的組換えを利用した、GATEWAY(登録商標;Life Technologies,Carlsbad,CA USA)クローニングなどを好適に例示することができる。 Recombinant cloning techniques that can be used to construct KCNJ2 gene-introduced adenovirus vectors include GATEWAY (registered) using site cloning of lambda phage in E. coli in addition to TA cloning and cloning using restriction enzymes. (Trademark: Life Technologies, Carlsbad, CA USA) cloning and the like can be preferably exemplified.
KCNJ2遺伝子の発現は、ウイルスベクターに組み込まれたGFP発現マーカーを蛍光顕微鏡、共焦点レーザー顕微鏡などで検鏡し、視覚的に確認することができる。他にも、市販又は定法により作製した抗KCNJ2抗体を用いたウェスタンブロット法、フローサイトメトリー法、免疫組織化学染色法、免疫蛍光染色法、ノーザンブロット法、RT−PCR法などにより定性・定量的に、KCNJ2遺伝子発現を測定することができる。 The expression of the KCNJ2 gene can be visually confirmed by examining a GFP expression marker incorporated in a viral vector with a fluorescence microscope, a confocal laser microscope or the like. Besides, qualitative and quantitative by Western blotting, flow cytometry method, immunohistochemical staining method, immunofluorescence staining method, Northern blotting method, RT-PCR method, etc. using commercially available or routinely prepared anti-KCNJ2 antibody In addition, KCNJ2 gene expression can be measured.
本発明における細胞内への遺伝子導入方法としては、KCNJ2遺伝子を細胞内に導入する限り特に制限されず、KCNJ2遺伝子を細胞内に導入する方法としては、リポソーム法、リポフェクション法、マイクロインジェクション法、DEAEデキストラン法、リン酸カルシウム法、エレクトロポレーション法等を挙げることができ、Lipofectin Reagent(登録商標)、Lipofectamine(登録商標)、Lipofectamine(登録商標)2000 Reagent(Invitrogen社製)や、SuperFect(登録商標)Transfection Reagent(キアゲン社製)、FuGENE(登録商標)HD Transfection Reagent(ロシュ・ダイアグノスティックス社製)、FuGENE(登録商標)6 Transfection Reagent(ロシュ・ダイアグノスティックス社製)等の市販のトランスフェクション試薬を用いる当技術分野で広く用いられている手法を挙げることができる。 The method for introducing a gene into a cell in the present invention is not particularly limited as long as the KCNJ2 gene is introduced into the cell. Examples of the method for introducing the KCNJ2 gene into the cell include a liposome method, a lipofection method, a microinjection method, DEAE. Dextran method, calcium phosphate method, electroporation method, and the like. Lipofectin Reagent (registered trademark), Lipofectamine (registered trademark), Lipofectamine (registered trademark) 2000 Reagent (manufactured by Invitrogen), SuperFect (registered trademark) Transfection Commercial transfections such as Reagent (Qiagen), FuGENE (registered trademark) HD Transfection Reagent (Roche Diagnostics), FuGENE (registered trademark) 6 Transfection Reagent (Roche Diagnostics) A technique widely used in the art using reagents. It can gel.
本発明におけるウイルスベクターに感染させる工程としては、iPS細胞から分化した心筋細胞及び心筋前駆細胞を含む胚様体若しくはコロニーを単一細胞に分離し、分離から12時間以内、より好ましくは6時間以内、特に好ましくは3時間以内、最も好ましくは1時間以内にウイルスベクターへの感染を開始させ、感染のインキュベーション時間としては、96時間、より好ましくは72時間、特に好ましくは48時間、最も好ましくは24時間のインキュベーション時間を例示することができる。 In the step of infecting with the viral vector in the present invention, an embryoid body or colony containing cardiomyocytes differentiated from iPS cells and myocardial progenitor cells is separated into single cells, and within 12 hours, more preferably within 6 hours after separation. Particularly preferably, the infection with the viral vector is started within 3 hours, most preferably within 1 hour, and the incubation time for infection is 96 hours, more preferably 72 hours, particularly preferably 48 hours, most preferably 24 hours. Time incubation time can be illustrated.
また、ウイルスベクターの感染に際しては、5000MOI、より好ましくは1000MOI、特に好ましくは500MOI、最も好ましくは100MOIの濃度で使用する。 For infection with a viral vector, it is used at a concentration of 5000 MOI, more preferably 1000 MOI, particularly preferably 500 MOI, and most preferably 100 MOI.
本発明における自発的な周期的収縮活性とは、細胞の周期的な電気的活動により、細胞が自発的かつ規則正しく収縮と弛緩を繰り返す現象をいい、Kir2.1チャネルを発現していないiPS細胞由来の心筋細胞は、この自発的な周期的収縮活性を有している。これに対して、本発明の心筋モデル細胞は自発的な周期的収縮活性を有しておらず、自発的ではなく(電気刺激を受けた場合に)細胞が規則正しく収縮と弛緩を繰り返す。細胞の自発的な周期的収縮活性は、目視観察によって判定することができ、ビデオカメラ又は顕微鏡などを用いて計数した、1視野あたりの自律拍動細胞数を、1視野あたりの細胞総数で標準化した、自律拍動細胞数率を用いて評価することができる。 The spontaneous periodic contractile activity in the present invention refers to a phenomenon in which a cell repeats contraction and relaxation spontaneously and regularly due to the periodic electrical activity of the cell, and is derived from an iPS cell not expressing the Kir2.1 channel. Cardiomyocytes have this spontaneous cyclic contractile activity. In contrast, the myocardial model cells of the present invention do not have spontaneous periodic contractile activity, and are not spontaneous (when subjected to electrical stimulation), the cells regularly contract and relax. Spontaneous periodic contractile activity of cells can be determined by visual observation, and the number of autonomous beating cells per visual field counted using a video camera or a microscope is standardized by the total number of cells per visual field. The autonomous pulsation cell count rate can be used for evaluation.
本発明における活動電位とは、ナトリウムイオン及びカリウムイオンを主体とする電荷をもつイオンが、細胞内外の濃度差に従い、イオンチャネルを通じて受動的に拡散を起こすことにより生じる、一過性の電位変化をいう。また、本発明における膜電位とは、上記イオンの濃度差により、細胞の内外で生じている電位の差をいい、静止膜電位とは、細胞内外を移動するイオンの流出入電荷量が等しい状態における、安定した膜電位をいい、陰性の電位である。 The action potential in the present invention is a transient potential change caused by passive diffusion of ions having charges mainly composed of sodium ions and potassium ions through an ion channel according to a concentration difference between inside and outside the cell. Say. In addition, the membrane potential in the present invention refers to the difference in potential generated inside and outside the cell due to the above-described difference in ion concentration, and the resting membrane potential is a state in which the inflow and outflow charges of ions moving inside and outside the cell are equal. The stable membrane potential is a negative potential.
本発明における最大拡張期電位とは、陰性の静止膜電位が、刺激により陽性電位へと入れ替わる脱分極を経て、もとの静止膜電位へ戻る再分極の過程における陰性頂点にあたる電位をいう。本発明の心筋モデル細胞の最大拡張期電位は、ヒト心室筋型心筋細胞に代表される細胞における最大拡張期電位と同程度の−85〜−65mVであることが好ましい。 The maximum diastolic potential in the present invention refers to a potential corresponding to a negative apex in a repolarization process in which a negative resting membrane potential returns to the original resting membrane potential through depolarization that is switched to a positive potential by stimulation. The maximum diastolic potential of the myocardial model cell of the present invention is preferably −85 to −65 mV, which is about the same as the maximum diastolic potential in cells represented by human ventricular myocardial cells.
本発明において、正常な内向きのカリウム電流における内向きのカリウム電流とは、静止膜電位の維持、及び、活動電位最終局面での静止膜電位への再分極化を司るIK1電流をいい、正常な内向きのカリウム電流特性とは、ヒト心筋細胞に代表される細胞が発生するIK1電流強度に比して、同程度のIK1電流強度を有する性質をいう。 In the present invention, the inward potassium current in normal inward potassium current refers to the IK1 current that governs the maintenance of the resting membrane potential and the repolarization to the resting membrane potential in the final action potential phase. The inward potassium current characteristic means a property having an IK1 current intensity comparable to the IK1 current intensity generated by cells typified by human cardiomyocytes.
本発明における活動電位測定の実施には、本発明の心筋モデル細胞の作製方法における工程(d)のウイルス感染後3日以降、より好ましくは3日以降30日以内、最も好ましくは14日以降30日以内の期間、培養した細胞を使用し、測定用の細胞外液・細胞内液バッファーとしては、pH6〜8のリン酸バッファー、トリス塩酸バッファーなどの、IK1電流に影響を与えないバッファーであれば特に制限されず、132mM NaCl,2mM CaCl2,10mM HEPES,4.8mM KCl,1.2mM MgCl2,5mM Glucoseの組成により調製されたバッファーを、NaOHによりpH7.4に調製した細胞外液や、110mM K-Aspartate,5mM ATP−K2,11mM EGTA,10mM HEPES,1mM CaCl2,1mM MgCl2の組成により調製されたバッファーを、KOHによりpH7.3に調製した細胞内液を、好ましく挙げることができる。For the action potential measurement in the present invention, 3 days after virus infection in step (d) in the method for producing myocardial model cells of the present invention, more preferably 3 days to 30 days, and most preferably 14 days to 30 days. Cells that have been cultured for a period of less than a day, and the extracellular / intracellular fluid buffer for measurement should be a buffer that does not affect the IK1 current, such as pH 6-8 phosphate buffer or Tris-HCl buffer. There is no particular limitation, and an extracellular solution prepared with a pH of 7.4 using NaOH prepared from a composition of 132 mM NaCl, 2 mM CaCl 2 , 10 mM HEPES, 4.8 mM KCl, 1.2 mM MgCl 2 , 5 mM Glucose 110 mM K-Aspartate, 5 mM ATP-K2, 11 mM EGTA, 10 mM HEPES, 1 mM CaC 2, a 1 mM MgCl 2 buffer prepared by the composition, intracellular solution prepared in pH7.3 by KOH, can be preferably exemplified.
本発明における活動電位、静止膜電位、最大拡張期電位測定を記録するには、パッチクランプ法又は全自動ハイスループットパッチクランプシステムを用いることができる。静止状態にある、本発明のKir2.1チャネル発現細胞における活動電位は、種々の保持電位及び脱分極刺激(コマンドパルス)を細胞に与えることにより誘発することができる。これらの条件については、当業者であれば容易に設定することができ、一例としては、0.5秒間のコマンドパルスを0.1Hzの周波数内で、−40mVから−150mVまで10mV刻みで細胞に与えることにより、誘発することができる。IK1電流成分の特定には、0.2mMのBaCl2を添加した細胞外液下で観察されるトレースを、BaCl2添加前のトレースから差し引かれた差分により同定される、Ba2+差分電流の値を用いることができる。To record the action potential, resting membrane potential, and maximum diastolic potential measurement in the present invention, the patch clamp method or a fully automatic high throughput patch clamp system can be used. Action potentials in Kir2.1 channel-expressing cells of the invention that are in a quiescent state can be triggered by applying various holding potentials and depolarizing stimuli (command pulses) to the cells. These conditions can be easily set by those skilled in the art. For example, a command pulse of 0.5 seconds is applied to a cell at a frequency of 0.1 Hz from −40 mV to −150 mV in 10 mV increments. It can be triggered by giving. For the identification of the IK1 current component, the value of Ba 2+ differential current, which is identified by the difference subtracted from the trace before addition of BaCl 2 , is observed in the extracellular solution supplemented with 0.2 mM BaCl 2. Can be used.
本発明の心筋細胞に対して毒性作用及び/又は変調作用を有する物質のスクリーニング方法としては、上記本発明の心筋モデル細胞と被検物質とを接触させる工程(A);電気生理学的試験手法を用いて、被検物質に起因する心筋細胞に対する毒性作用及び/又は変調作用を検出する工程(B);工程(B)の検出結果に基づき、被検物質の心筋細胞に対する毒性作用及び/又は変調作用の有無を判定する工程(C);の各工程を備えた方法であれば特に制限されず、上記工程(B)における毒性作用及び/又は変調作用が、hERG電流阻害活性であることが好ましい。 The method for screening a substance having a toxic and / or modulating action on the cardiomyocytes of the present invention includes the step (A) of bringing the myocardial model cell of the present invention into contact with a test substance; A step (B) of detecting a toxic effect and / or modulation effect on cardiomyocytes caused by the test substance; a toxic action and / or modulation of the test substance on cardiomyocytes based on the detection result of step (B) Step (C) for determining the presence or absence of action is not particularly limited as long as it is a method comprising each step, and the toxic action and / or modulation action in step (B) is preferably hERG current inhibitory activity. .
例えば、電気生理学的試験手法を用いて、被検物質に起因する心筋細胞に対する毒性作用や変調作用を検出する方法としては、全自動ハイスループットパッチクランプシステムを含むパッチクランプ法、アイソトープ及び原始吸光分析によるイオンフラックス法、膜電位感受性色素法、イオン感受性蛍光色素法、多点平面電極システムを利用した細胞外電位測定法、MEA(Multielectrode Array)システムによる多電極記録法、FRET等の分子プローブを含む膜電位感受性色素を用いた活動電位測定法、膜電位イメージング法、イオン流束(Ion flux)解析法、放射性リガンド結合試験法などを好適に挙げることができる。 For example, methods for detecting toxic effects and modulation effects on cardiomyocytes caused by test substances using electrophysiological testing techniques include patch clamp methods including fully automatic high-throughput patch clamp systems, isotopes and primitive absorption analysis Includes ion-flux method, membrane potential-sensitive dye method, ion-sensitive fluorescent dye method, extracellular potential measurement method using multi-point planar electrode system, multi-electrode recording method using MEA (Multielectrode Array) system, and molecular probes such as FRET Preferred examples include an action potential measurement method using a membrane potential sensitive dye, a membrane potential imaging method, an ion flux analysis method, and a radioligand binding test method.
上記毒性作用や変調作用は、hERG電流阻害活性、細胞機能への影響、マーカーの発現、受容体結合、電気刺激による収縮活性、電気生理学的特性変化等を検出することにより明らかにすることができる。上記hERG電流阻害活性を検出するには、本発明の心筋モデル細胞のQT間隔延長作用を評価すればよく、かかるQT間隔延長作用評価法としては、被検物質との接触により、活動電位持続時間(APD)や、MEAシステムにおけるフィールドポテンシャル持続時間(FPD)の延長を測定する方法を挙げることができる。FPDの測定によると、Naチャネルでのイオン流入による、脱分極の大きなピークを始点とし、カリウムチャネルによる再分極のピーク(2ndピーク)を終点として計測される値をもとに、被検物質によるQT延長作用を評価することができる。 The above toxic and modulation effects can be clarified by detecting hERG current inhibitory activity, effects on cell function, marker expression, receptor binding, contractile activity by electrical stimulation, changes in electrophysiological characteristics, etc. . In order to detect the hERG current inhibitory activity, the QT interval prolonging action of the myocardial model cell of the present invention may be evaluated. As such a QT interval prolonging action evaluation method, the action potential duration time is determined by contact with a test substance. (APD) and a method for measuring the extension of the field potential duration (FPD) in the MEA system. According to the FPD measurement, it depends on the test substance based on the values measured starting from the peak of depolarization due to ion inflow in the Na channel and starting from the peak of repolarization by the potassium channel (2nd peak). QT prolongation action can be evaluated.
上記本発明のスクリーニング方法は、創薬のスクリーニングに有用である。かかる創薬スクリーニングによると、被検物質の細胞機能への影響、マーカーの発現、受容体結合、電気刺激による収縮活性、電気生理学的特性変化の解析などにより、副作用のない薬剤としての有効性を評価することができる。影響が観察される場合、被検物質の濃度を力価測定し、致死量及び半有効量を決定することができる。 The screening method of the present invention is useful for drug discovery screening. According to this drug discovery screening, the effects of test substances on cell function, marker expression, receptor binding, contractile activity by electrical stimulation, analysis of changes in electrophysiological properties, etc., have proved effective as drugs without side effects. Can be evaluated. If an effect is observed, the concentration of the test substance can be titrated to determine the lethal dose and semi-effective dose.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
1.hiPS細胞の培養
hiPS細胞株(201B7,253G1)は、Basic fibroblast growth factor(10ng/ml)(bFGF;R&D Systems)を加えた霊長類ES細胞培養液(ReproCell)中で、1mg/mlのmitomycin C solution(Nacalai Tesque)で処理されたSNL76/7(European Collection of Cell Culture)の支持細胞層上に播種し、培養を行った。継代時は、hiPS細胞コロニーを酵素処理により単一細胞化し、支持細胞でコーティングされた培養ディッシュに撒き、継代培養を行った。
2.心筋細胞への分化誘導
心筋細胞への分化誘導は過去の報告(PLoS ONE 6(8):e23657,2011)を、若干改変した手法で行った。共培養した支持細胞を除くために、iPS細胞はmTeSR1培養液(Stem cell technologies)中のmatrigel(Invitrogen)基底膜マトリックス上で数世代継代し、分化誘導の前日に、1:60希釈にしたmatrigelで細胞を覆った。心筋細胞への分化誘導するため、mTeSR1培養液を100ng/mlのActivin A(R&D Systems)を加えたB27加RPMI1640培養液(RPMI/B27)に換えて24時間培養し、続いてbonemorphogenetic protein 4(10ng/ml, R&D Systems)とbFGF(10ng/ml)を加えて4日間培養した。5日目に、培養液をRPMI/B27/Dkk1(100ng/ml,R&D Systems)に換えた後、7日目にはRPMI/B27に換え、2−3日ごとに培養液の交換を行った。9日目には自律拍動する細胞が観察された。
3.分化済み心筋細胞の前培養
分化済みのhiPS細胞由来心筋細胞として市販されている、iCell-cardiomyocytes(iCell−CMs)(Cellular Dynamics International;CDI,Madison,WI,USA)を、iPS Academia Japan Inc.社(Kyoto,Japan)より購入した。iCell−CMsについて、製造元(CDI)より提供されている方法に従い解凍し、アデノウイルスに感染させるまでの4日間、前培養を行った。ヒトES細胞由来心筋細胞の胚様体は、Cellectis社(Sweden)より購入し、酵素処理により単一細胞化した後、製造元(Cellectis)より提供されている方法に従いアデノウイルスに感染させるまでの4日間、前培養を行った。
4.KCNJ2発現プラスミドベクターの作製
KCNJ2−EGFP融合タンパク質の遺伝子をコードする、プラスミドのpENTR−KCNJ2−EGFPについて、cagcttgccgtctctcatgg (KCNJ2 reverse)[配列番号3]、gtccccaacactcccctttg (KCNJ2 forward) [配列番号4]、cgtctccgtccagctcgaccag (EGFP reverse)[配列番号5]、gaccacatgaagcagcacgac (EGFP forward)[配列番号6]、の4プライマーにより全長の翻訳領域の配列を決定した。全長翻訳領域配列は、制限酵素のSalIとNotIで切断され、T4 DNA ligase(Takara)のライゲーションにより、pENTR1Aベクター(Invitrogen)へ配列を挿入した。LR Clonase II Enzyme Mix(Invitrogen)のLR反応により、エントリークローンであるpENTR1A−KCNJ2−EGFPを介した、pAd/CMV/V5−DEST(Invitrogen)へのターゲット遺伝子組み換えを行った。得られた発現クローンのpAd−KCNJ2−EGFPを増幅させ、純正プラスミドDNAを精製した。HEK293A細胞へ形質導入する前に、左右両端の逆方向反復配列(ITR又はパリンドローム配列)を、Lipofectamine(登録商標)2000 Reagent (Invitrogen)に曝露させた。アデノウイルス発現クローンを増幅するHEK293A細胞は、定常的にE1タンパクを発現しており、HEK細胞及びiCell−CMsへの遺伝子導入用に回収して用いた。
5.心筋細胞へのアデノウイルスを介した遺伝子導入
前培養した幹細胞由来心筋細胞を、分散・分離溶液のアキュターゼを用いた酵素処理により単一細胞化し、lamimin/poly-D/L-lysineコーティングされたガラスボトムディッシュに撒いた。心筋細胞をアデノウイルスに感染させるために、100MOIの濃度のpAd−EGFP又はpAd−KCNJ2−EGFPに、24時間曝露させた。感染開始から48時間後の感染率(心筋細胞におけるGFPシグナル陽性率)は、60〜100%(平均約80%)であった。アデノウイルス感染心筋細胞は、10%FBS、1mM GlutaMAX(Invitrogen)、0.1mM non essential amino acid(Sigma)、0.1mM 2-mercapto ethanol(Sigma)加Knockout DMEM(Invitrogen)下にて培養を行った。培養細胞は、アデノウイルスによる遺伝子導入完了後30日の間は、培養により維持が可能であり、以下の解析には全て導入から30日までの感染細胞を用いて実施した。膜電流及び活動電位測定は、全てGFP陽性細胞を用いて行った。同様に、HEK293細胞への遺伝子導入用として、HEK293細胞をDulbecco's Modified Eagle Medium(Gibco)で培養し、100MOI(Multiplicity Of Infection)の濃度のpAd−EGFP又はpAd−KCNJ2−EGFPに感染させた。KCNJ2遺伝子の発現から48時間後、GFP陽性細胞における膜電流を測定した。
6.免疫細胞染色及び画像解析
解析に用いた細胞は、全てlamimin/poly-D/L-lysineコーティングされたガラスボトムディッシュ又はチャンバー上に培養し、免疫細胞染色には、氷冷した100%エタノールに5〜15分置いて固定・浸透化させた。また、GFP画像解析には、4%パラホルムアルデヒドの後、0.1% Triton-Xに浸透化させて用いた。引き続いて、phosphate-buffered saline(PBS)にて洗浄し、免疫細胞染色には、5% fetal bovine serum/PBSにて30分間ブロッキングを行い、1次抗体との反応を4℃にて一晩行った後、Alexa 488又はAlexa 647にて標識された2次抗体イムノグロブリンGとの反応を、室温にて2時間行った。1次抗体はAlexa 488又はAlexa 647標識2次抗体でラベルされ、余分な抗体はPBSにて洗浄した。画像解析に用いた全細胞は、PBSを取り除いた後、ガラスボトムディッシュ又はスライドガラス上で、VECTASHIELD mounting medium(Vector,Burlingame,USA)にて封入を行った。細胞の観察には、Zeiss LSM510共焦点レーザー倒立型顕微鏡を使用し、励起光と蛍光の波長をそれぞれEx; 488nm,Em;505〜520nmとEx;635nm,Em;650〜670nmに設定した100倍油浸対物レンズを用いるか、又は、Olympus IX-71倒立型蛍光顕微鏡を使用し、キセノンランプを(励起光:492±9nm、吸収フィルタ:530±18nm)に設定した、40倍油浸対物レンズを用いて行った。共焦点顕微鏡画像は、リニアレンジの蛍光強度/ピクセルを用いた光電子増倍管ゲインの自動調整により最適化され、16回の取り込み画像を平均化したものを採用した。
7.心筋細胞の収縮性解析
分化誘導された心筋細胞とそのクラスターの収縮活性の観察には、ビデオカメラ(Hitachi)又は顕微鏡(IX-71)を使用した。収縮率は、10−sビデオカメライメージ若しくは、1分間の目視観察により、収縮回数を計数して算出した。クラスターについては、GFP陽性細胞を3以上含むものを、収縮性解析に使用した。hiPS細胞から心筋細胞への分化誘導率を割り出すために、自律拍動する細胞数は、顕微鏡の1視野あたりの細胞総数で標準化した。IK1内向き整流K+電流を阻害するために、0.2mMのBa2+をバスへ投与した。静止状態のKCNJ2−GFP細胞クラスターには、電気刺激装置(8EN-7203,NIHON KOHDEN,Tokyo,Japan)を用いた10ms、50V/cm、1Hzの均一な電場による、長期的電位刺激を与えた。
8.電場電位解析
iPS細胞由来及びES細胞由来の心筋細胞における、自発的並びに誘発的な電場電位を計測するために、MED64 multi-electrode array system (Alpha MED Science,Osaka,Japan)を使用した。細胞は、MEDプローブに直接入れて培養し、非侵襲性平面微小電極を用いて自発的又は電気刺激誘発性の電場電位を調べた。電気刺激は、MEDプローブ上の、2つの隣接する電極から誘起させた。
9.運動ベクトル解析
自律拍動する心筋細胞の運動ベクトルは、[Ghanbari, M. The cross-search algorithm for motion estimation. IEEE Transactions on communications 38, 950-953 (1990)]及び[Hayakawa, T. et al. Noninvasive evaluation of contractile behavior of cardiomyocyte monolayers based on motion vector analysis. Tissue engineering. Part C, Methods 18, 21-32 (2012)]に記載の、ブロックマッチング・アルゴリズムを用いた解析により算出した。
10.パッチクランプ
活動電位と膜電流は、Axopatch 200Bパッチクランプ増幅器(Molecular Devices,CA,USA)を用いた穿孔パッチ立体構成のパッチクランプ法により記録した。電信情報は、5kHzのローパスフィルター設定を用いて2〜5kHzで検出した。pCLAMP software(version10.02,Axon)は、voltage-pulseプロトコール作成や、データの取得及び解析に使用した。阻害剤などは、バス溶液へ迅速灌流システムを用いて投与した。hiPS由来心筋細胞又はiCell心筋細胞(幹細胞由来心筋細胞)を用いた全ての解析は、36±1℃の環境下で実施した。また、同様にHEK293細胞においても、ホールセル・パッチクランプ技術によるIK1電流の測定を実施した。細胞外液は、132mM NaCl,2mM CaCl2,10mM HEPES,4.8mM KCl,1.2mM MgCl2,5mM Glucose(NaOHでpH7.4に調製)、細胞内液は、110mM K-Aspartate,5mM ATP-K2,11mM EGTA,10mM HEPES,1mM CaCl2,1mM MgCl2(KOHでpH7.3に調製)の組成で調製した。IK1電流と電流電圧曲線は、hiPS由来心筋細胞・HEK293細胞共に同一の電圧プロトコールで測定した。−100mVの電気刺激により誘発されたIK1電流は、全細胞破裂パッチクランプ(Whole-cell ruptured patch-clamp)法により、直列抵抗が3〜5MΩの状態で細胞膜が破裂した後から測定を開始し、IK1電流の振幅が安定するまで(〜2−3分間)測定を続けた。その後、電流電圧曲線の解析を行った。
11.パッチクランプ法による解析
幹細胞由来心筋細胞の解析には、細胞をlaminin/poly-D/L-lysineコーティングされたディッシュに撒いたものを、倒立型顕微鏡(IX-71,Olympus)のステージに設置し、培養液をTyrode’s solution(135mM NaCl/0.33mM NaH2PO4/5.4mM KCl/1.8mM CaCl2/0.53mM MgCl2/5.5mM Glucose/5mM HEPES,pH7.4)に置き換えた。組成が110mM aspartic acid,30mM KCl,1mM CaCl2,5mM adenosine-5’-triphosphate magnesium salt,5mM creatine phosphate disodium salt,5mM HEPES,10mM EGTA(pH7.25)の細胞内液で満たされた場合の、微小電極の尖端抵抗は1.5〜4MΩであった。パッチ穿孔(10〜20MΩ、直列抵抗)を得るために、amphotericin B(0.3−0.6μg/ml;Nacalai)を細胞内液に投与した。パッチピペット内の前面を細胞内液に浸して満たし、背面をamphotericin Bを添加した細胞内液で満たした。hiPS細胞由来心筋細胞(201B7)、iCell心筋細胞及びヒトES細胞由来心筋細胞の細胞膜静電容量はそれぞれ、49.8±8.1 pF(n=19)、46.2±2.7pF(n=74)、43.7±4.8pF(n=13)であった。
12.パッチクランプ法の測定条件
幹細胞由来心筋細胞の活動電位及び静止膜電位は、電流固定法を用いて記録した。パッチクランプに使用した細胞の評価基準は、結果の項目に記す。自律拍動性心筋細胞の活動電位は、電気刺激無しに記録した。特に別途記載のない限り、静止状態の心筋細胞については、持続時間が0.2〜3.1msの脱分極電流刺激で、かつ、電流強度が閾上であり(活動電位を誘発する最低値の120%)、周波数が1Hzの電流を流すことで、活動電位を誘発させた。IK1(Kir2.1)電流の電流電圧曲線を得るために、0.5秒間のコマンドパルスを0.1Hzの周波数内で、−40mVから−150mVまで10mV刻みで刺激した。IK1電流成分は、0.2mMのBaCl2を添加した細胞外液下で差し引かれたトレースにより同定される、Ba2+差分電流として示される。電流電圧の相関曲線は、コマンドパルスの電圧に対するBa2+差分電流のピーク振幅を求めることで得られる。
13.薬剤・化合物
選択的Ikr阻害薬のE4031は、エーザイ株式会社(Japan)から提供されたものを使用した。他の全ての化合物は、医薬品グレードのものを使用し、一般的な流通経路により入手した。200mM BaCl2(水溶液)、1M CsCl(水溶液)、10mM E4031(水溶液)のストック溶液は、パッチクランプの細胞外液中に用時、最終濃度に調製して使用した。E4031のストック溶液については、使用する日と同日に調製した。
14.RNA抽出及び、定量的real-time PCR
iPS細胞(201B7)又はiCell心筋細胞からの全RNA抽出には、RNeasy kit(Qiagen)を使用した。相補的DNAは、プライマーとしてランダムヘキサマー(Applied Biosystems)を用いた逆転写により作製した。定量的real-time PCRは、ABI7300 cyclerを用いて実施した。KCNJ2プライマー(sense primer; 5’-TGTCACGGATGAATGCCCAA-3’配列番号7, antisense primer; 5’-CAAACACAGCTTGCCGTCTC-3’配列番号8)によるKCNJ2発現の解析には、SYBR greenを使用した。これらの解析は、製造者によって推奨されるプロトコールに沿って実施した。SYBR greenを使用した場合、PCR産物は、常時、解離曲線解析ソフトウェア(Applied Biosystems)により確認作業を行った。転写量は、ΔΔCT法を用いて比較を行った。ハウスキーピング遺伝子のコントロールとして、GAPDHを用い、プライマーは(sense primer; 5’-GAGCCACATCGCTCAGACAC-3’ 配列番号9, antisense primer; 5’-CATGTAGTTGAGGTCAATGAAGG-3’ 配列番号10)を使用した。
15.データ解析・統計処理
全ての数値は、平均値±標準誤差で示す。pCLAMP software ver.9.2and10.3(Molecular Dynamics)は、パッチクランプでのデータ取得及び解析に使用した。活動電位パラメータの解析には、Peak Analysis Module搭載のLabchart ver.7.2(ADInstruments)を使用した。図表作成及び統計処理には、OriginPro9.0J(Microcal)、Illustrator CS6.0(Adobe)、Instat program(GraphPad)を使用した。特に記載の無い場合、2群比較ではステューデントt検定を、多重比較では分散分析とBonferroni検定を行い、統計的有意差を求めた。P値<0.05を統計的有意差有りとした。
16.エピソーマル型ベクターを使用した遺伝子導入
続いて、エピソーマル型ベクターを使用した遺伝子導入による成熟化心筋の作成を試みた。1. Culture of hiPS cells The hiPS cell line (201B7,253G1) was cultured in a primate ES cell culture solution (ReproCell) supplemented with basic fibroblast growth factor (10 ng / ml) (bFGF; R & D Systems) at 1 mg / ml mitomycin C. It seed | inoculated on the support cell layer of SNL76 / 7 (European Collection of Cell Culture) processed with solution (Nacalai Tesque), and culture | cultivated. At the time of subculture, the hiPS cell colonies were made into single cells by enzyme treatment, spread on a culture dish coated with feeder cells, and subculture was performed.
2. Differentiation induction into cardiomyocytes Differentiation induction into cardiomyocytes was performed by a slightly modified technique from the previous report (PLoS ONE 6 (8): e23657,2011). To remove co-cultured feeder cells, iPS cells were passaged for several generations on a matrigel (Invitrogen) basement membrane matrix in mTeSR1 medium (Stem cell technologies) and diluted 1:60 the day before differentiation induction. The cells were covered with matrigel. In order to induce differentiation into cardiomyocytes, the mTeSR1 culture medium was replaced with B27-added RPMI1640 culture medium (RPMI / B27) supplemented with 100 ng / ml of Activin A (R & D Systems) and cultured for 24 hours, followed by bonemorphogenetic protein 4 ( 10 ng / ml, R & D Systems) and bFGF (10 ng / ml) were added and cultured for 4 days. On the fifth day, after changing the culture solution to RPMI / B27 / Dkk1 (100 ng / ml, R & D Systems), on the seventh day, it was changed to RPMI / B27, and the culture solution was changed every 2-3 days. . On the 9th day, autonomously pulsating cells were observed.
3. Pre-culture of differentiated cardiomyocytes iCell-cardiomyocytes (iCell-CMs) (Cellular Dynamics International; CDI, Madison, WI, USA), which are commercially available as differentiated hiPS cell-derived cardiomyocytes, are obtained from iPS Academia Japan Inc. (Kyoto, Japan). iCell-CMs were thawed according to the method provided by the manufacturer (CDI) and pre-cultured for 4 days until adenovirus infection. The embryoid body of human ES cell-derived cardiomyocytes is purchased from Cellectis (Sweden), converted into a single cell by enzymatic treatment, and then infected with adenovirus according to the method provided by the manufacturer (Cellectis). Pre-culture was performed for days.
4). Preparation of KCNJ2 expression plasmid vector For the plasmid pENTR-KCNJ2-EGFP, which encodes the gene for KCNJ2-EGFP fusion protein, cagcttgccgtctctcatgg (KCNJ2 reverse) [SEQ ID NO: 3], gtccccaacactcccctttg (KCNJ2 forward) [SEQ ID NO: 4 The sequence of the full-length translation region was determined with 4 primers of EGFP reverse) [SEQ ID NO: 5] and gaccacatgaagcagcacgac (EGFP forward) [SEQ ID NO: 6]. The full-length translation region sequence was cleaved with restriction enzymes SalI and NotI, and the sequence was inserted into the pENTR1A vector (Invitrogen) by ligation with T4 DNA ligase (Takara). By LR reaction of LR Clonase II Enzyme Mix (Invitrogen), target gene recombination into pAd / CMV / V5-DEST (Invitrogen) was performed via the entry clone pENTR1A-KCNJ2-EGFP. The resulting expression clone, pAd-KCNJ2-EGFP, was amplified, and pure plasmid DNA was purified. Prior to transduction into HEK293A cells, inverted repeats (ITR or palindromic sequences) at both ends were exposed to Lipofectamine® 2000 Reagent (Invitrogen). HEK293A cells that amplify adenovirus expression clones constantly express E1 protein, and were collected and used for gene transfer into HEK cells and iCell-CMs.
5). Adenovirus-mediated gene introduction into cardiomyocytes Pre-cultured stem cell-derived cardiomyocytes are made into single cells by enzymatic treatment using a dispersion / separation solution of acactase, and laminin / poly-D / L-lysine-coated glass I went to the bottom dish. To infect myocardial cells with adenovirus, they were exposed to pAd-EGFP or pAd-KCNJ2-EGFP at a concentration of 100 MOI for 24 hours. The infection rate (GFP signal positive rate in cardiomyocytes) 48 hours after the start of infection was 60 to 100% (average of about 80%). Adenovirus-infected cardiomyocytes are cultured in Knockout DMEM (Invitrogen) with 10% FBS, 1 mM GlutaMAX (Invitrogen), 0.1 mM non essential amino acid (Sigma), and 0.1 mM 2-mercapto ethanol (Sigma). It was. The cultured cells can be maintained by culturing for 30 days after completion of gene introduction by adenovirus, and all the following analyzes were performed using infected cells from the introduction to 30 days. Membrane current and action potential measurements were all performed using GFP positive cells. Similarly, for gene introduction into HEK293 cells, HEK293 cells were cultured in Dulbecco's Modified Eagle Medium (Gibco) and infected with pAd-EGFP or pAd-KCNJ2-EGFP at a concentration of 100 MOI (Multiplicity Of Infection). 48 hours after the expression of the KCNJ2 gene, the membrane current in GFP positive cells was measured.
6). Immune cell staining and image analysis All the cells used for analysis were cultured on a glass bottom dish or chamber coated with laminin / poly-D / L-lysine. For immune cell staining, 5% in ice-cold 100% ethanol was used. ˜15 minutes to fix and permeabilize. For GFP image analysis, 4% paraformaldehyde was used and then permeabilized with 0.1% Triton-X. Subsequently, the cells were washed with phosphate-buffered saline (PBS), and for immune cell staining, blocked with 5% fetal bovine serum / PBS for 30 minutes and reacted with the primary antibody at 4 ° C overnight. Thereafter, the reaction with the secondary antibody immunoglobulin G labeled with Alexa 488 or Alexa 647 was carried out at room temperature for 2 hours. The primary antibody was labeled with Alexa 488 or Alexa 647 labeled secondary antibody, and excess antibody was washed with PBS. All cells used for image analysis were sealed with VECTASHIELD mounting medium (Vector, Burlingame, USA) on a glass bottom dish or slide glass after removing PBS. For observation of cells, Zeiss LSM510 confocal laser inverted microscope was used, and the wavelengths of excitation light and fluorescence were set to Ex; 488 nm, Em; 505-520 nm and Ex; 635 nm, Em; 650-670 nm, respectively. 40x oil immersion objective lens using an oil immersion objective lens or using an Olympus IX-71 inverted fluorescence microscope and setting a xenon lamp (excitation light: 492 ± 9 nm, absorption filter: 530 ± 18 nm) It was performed using. The confocal microscope image was optimized by automatic adjustment of the photomultiplier tube gain using a linear range of fluorescence intensity / pixel, and an average of 16 captured images was used.
7). Analysis of contractility of cardiomyocytes A video camera (Hitachi) or a microscope (IX-71) was used to observe the contractile activity of differentiated cardiomyocytes and their clusters. The contraction rate was calculated by counting the number of contractions by 10-s video camera image or visual observation for 1 minute. As for clusters, those containing 3 or more GFP positive cells were used for contractility analysis. In order to determine the differentiation induction rate from hiPS cells to cardiomyocytes, the number of cells that autonomously beat was standardized by the total number of cells per field of view of the microscope. To inhibit the IK1 inward rectifier K + current, 0.2 mM Ba 2+ was administered to the bath. The stationary KCNJ2-GFP cell cluster was subjected to long-term potential stimulation with a uniform electric field of 10 ms, 50 V / cm, 1 Hz using an electrical stimulator (8EN-7203, NIHON KOHDEN, Tokyo, Japan).
8). Electric field potential analysis The MED64 multi-electrode array system (Alpha MED Science, Osaka, Japan) was used to measure spontaneous and induced electric field potentials in iPS cell-derived and ES cell-derived cardiomyocytes. Cells were cultured directly in MED probes and examined for spontaneous or electrical stimulation-induced field potentials using non-invasive planar microelectrodes. Electrical stimulation was induced from two adjacent electrodes on the MED probe.
9. Motion vector analysis The motion vectors of autonomously pulsating cardiomyocytes are described in [Ghanbari, M. The cross-search algorithm for motion estimation. IEEE Transactions on communications 38, 950-953 (1990)] and [Hayakawa, T. et al. Noninvasive evaluation of contractile behavior of cardiomyocyte monolayers based on motion vector analysis. Tissue engineering. Part C, Methods 18, 21-32 (2012)] was calculated by analysis using a block matching algorithm.
10. Patch Clamp Action potentials and membrane currents were recorded by a perforated patch configuration patch clamp method using an Axopatch 200B patch clamp amplifier (Molecular Devices, CA, USA). Telegraph information was detected at 2-5 kHz using a 5 kHz low pass filter setting. pCLAMP software (version 10.02, Axon) was used for voltage-pulse protocol creation, data acquisition and analysis. Inhibitors and the like were administered to the bath solution using a rapid perfusion system. All analyzes using hiPS-derived cardiomyocytes or iCell cardiomyocytes (stem cell-derived cardiomyocytes) were performed in an environment of 36 ± 1 ° C. Similarly, HEK293 cells were measured for IK1 current using the whole cell patch clamp technique. The extracellular fluid is 132 mM NaCl, 2 mM CaCl 2 , 10 mM HEPES, 4.8 mM KCl, 1.2 mM MgCl 2 , 5 mM Glucose (adjusted to pH 7.4 with NaOH), and the intracellular fluid is 110 mM K-Aspartate, 5 mM ATP. -K2,11mM EGTA, 10mM HEPES, were prepared with the composition of 1mM CaCl 2, 1mM MgCl 2 ( KOH in prepared pH 7.3). The IK1 current and the current-voltage curve were measured using the same voltage protocol for both hiPS-derived cardiomyocytes and HEK293 cells. The IK1 current induced by -100 mV electrical stimulation is measured after the cell membrane is ruptured with a series resistance of 3 to 5 MΩ by the whole-cell ruptured patch-clamp method. The measurement was continued until the amplitude of the IK1 current stabilized (˜2-3 minutes). Thereafter, the current-voltage curve was analyzed.
11. Analysis by patch clamp method For analysis of stem cell-derived cardiomyocytes, a cell is spread on a laminin / poly-D / L-lysine-coated dish and placed on the stage of an inverted microscope (IX-71, Olympus). The culture solution was replaced with Tyrode's solution (135 mM NaCl / 0.33 mM NaH 2 PO 4 /5.4 mM KCl / 1.8 mM CaCl 2 /0.53 mM MgCl 2 /5.5 mM Glucose / 5 mM HEPES, pH 7.4). . When the composition is filled with an intracellular solution of 110 mM aspartic acid, 30 mM KCl, 1 mM CaCl 2 , 5 mM adenosine-5′-triphosphate magnesium salt, 5 mM creatine phosphate disodium salt, 5 mM HEPES, 10 mM EGTA (pH 7.25), The tip resistance of the microelectrode was 1.5-4 MΩ. In order to obtain patch perforations (10-20 MΩ, series resistance), amphotericin B (0.3-0.6 μg / ml; Nacalai) was administered to the intracellular fluid. The front surface in the patch pipette was filled with intracellular fluid and filled, and the back surface was filled with intracellular fluid to which amphotericin B was added. The cell membrane capacitances of hiPS cell-derived cardiomyocytes (201B7), iCell cardiomyocytes and human ES cell-derived cardiomyocytes were 49.8 ± 8.1 pF (n = 19) and 46.2 ± 2.7 pF (n, respectively). = 74), 43.7 ± 4.8 pF (n = 13).
12 Measurement conditions of patch clamp method The action potential and resting membrane potential of stem cell-derived cardiomyocytes were recorded using the current clamp method. The evaluation criteria of the cells used for the patch clamp are described in the result item. The action potential of autonomic pulsatile cardiomyocytes was recorded without electrical stimulation. Unless otherwise stated, quiescent cardiomyocytes are depolarized current stimuli with a duration of 0.2-3.1 ms and the current intensity is above the threshold (the lowest value that induces action potentials). 120%), an action potential was induced by passing a current with a frequency of 1 Hz. In order to obtain a current-voltage curve of the IK1 (Kir2.1) current, a 0.5 second command pulse was stimulated from −40 mV to −150 mV in 10 mV increments within a frequency of 0.1 Hz. The IK1 current component is shown as Ba 2+ differential current, identified by traces subtracted under extracellular fluid supplemented with 0.2 mM BaCl 2 . The correlation curve of the current voltage is obtained by calculating the peak amplitude of the Ba 2+ differential current with respect to the command pulse voltage.
13. Drug / Compound Selective Ikr inhibitor E4031 used was provided by Eisai Co., Ltd. (Japan). All other compounds used were pharmaceutical grade and were obtained by general distribution channels. A stock solution of 200 mM BaCl 2 (aqueous solution), 1M CsCl (aqueous solution), and 10 mM E4031 (aqueous solution) was used at the final concentration when used in the extracellular solution of the patch clamp. A stock solution of E4031 was prepared on the same day of use.
14 RNA extraction and quantitative real-time PCR
RNeasy kit (Qiagen) was used for total RNA extraction from iPS cells (201B7) or iCell cardiomyocytes. Complementary DNA was prepared by reverse transcription using random hexamers (Applied Biosystems) as primers. Quantitative real-time PCR was performed using ABI7300 cycler. SYBR green was used for the analysis of KCNJ2 expression by the KCNJ2 primer (sense primer; 5′-TGTCACGGATGAATGCCCAA-3 ′ SEQ ID NO: 7, antisense primer; 5′-CAAACACAGCTTGCCGTCTC-3 ′ SEQ ID NO: 8). These analyzes were performed according to the protocol recommended by the manufacturer. When SYBR green was used, the PCR product was always checked with dissociation curve analysis software (Applied Biosystems). The amount of transfer was compared using the ΔΔCT method. GAPDH was used as a housekeeping gene control, and primers (sense primer; 5′-GAGCCACATCGCTCAGACAC-3 ′ SEQ ID NO: 9, antisense primer; 5′-CATGTAGTTGAGGTCAATGAAGG-3 ′ SEQ ID NO: 10) were used.
15. Data analysis / statistical processing All numerical values are shown as mean ± standard error. pCLAMP software ver.9.2and10.3 (Molecular Dynamics) was used for data acquisition and analysis with patch clamp. Labchart ver.7.2 (ADInstruments) equipped with Peak Analysis Module was used for analysis of action potential parameters. OriginPro9.0J (Microcal), Illustrator CS6.0 (Adobe), and Instat program (GraphPad) were used for chart creation and statistical processing. Unless otherwise specified, Student t-test was performed for two-group comparisons, and analysis of variance and Bonferroni test were performed for multiple comparisons to obtain statistical significance. P value <0.05 was considered statistically significant.
16. Gene transfer using episomal vector Subsequently, an attempt was made to create a matured myocardium by gene transfer using an episomal vector.
pIRES2-EGFP-KCNJ2(Hosaka et al. JMCC 2003 35:409-15)からSalI/NotIもしくはSalI/BamHIで切りだしたヒトKCNJ2を含むインサートをそれぞれの制限酵素のセットでカットしたpEBMulti-Neoベクター(和光純薬)にサブクローニングした。図7に示すように、SalI/NotIで切りだしたインサートには、マーカーとしてEGFP蛋白が発現し、SalI/BamHIで切りだしたインサートはhKCNJ2クローンのみが含まれる。遺伝子導入の効果を調べる際には、EGFPを標識して行った。 pEBMulti-Neo vector obtained by cutting the insert containing human KCNJ2 cut with SalI / NotI or SalI / BamHI from pIRES2-EGFP-KCNJ2 (Hosaka et al. JMCC 2003 35: 409-15) with each restriction enzyme set ( Subcloned into Wako Pure Chemicals). As shown in FIG. 7, the insert cut out with SalI / NotI expresses EGFP protein as a marker, and the insert cut out with SalI / BamHI contains only the hKCNJ2 clone. When examining the effect of gene transfer, EGFP was labeled.
iCell-CM(CDI社)は、CDI社のプロトコルとNakamura et al. J Pharmacol Sci 124:494-501 (2014)に従い、凍結バイアルから解凍し、35 mmもしくは60 mmプラスチックディッシュ上で前培養を行った。培地を始め試薬類は全て細胞に同封されるキットのものを使用した。初回解凍後は、バイアル当たり6 cmディッシュ3枚の底面積を基準にして単層培養を行い、2−7日後の植え換えの際に測定系に応じた処理を施した。 iCell-CM (CDI) is thawed from frozen vials according to CDI protocol and Nakamura et al. J Pharmacol Sci 124: 494-501 (2014) and pre-cultured on 35 mm or 60 mm plastic dishes. It was. All the reagents including the medium were from the kit enclosed with the cells. After the first thawing, monolayer culture was performed on the basis of the bottom area of three 6 cm dishes per vial, and treatment according to the measurement system was performed at the time of replanting 2-7 days later.
遺伝子をトランスフェクションする場合の細胞単離法について次に示す。 The cell isolation method for gene transfection is described below.
細胞単離法(再播種);実験プロトコルは下記に示すものであった。
・37℃に温めたPBS(-)で3回洗う。
・37℃に温めた0.5 mL accutase (innovative cell technologies)を与え、CO2インキュベーターで 37 ℃ 30分静置する。
・30-50 %の細胞が丸くなりディッシュの底から浮いていることを確認し、ディッシュをそっと傾けて混ぜると、9割の細胞を底から浮かせる。
・37℃に温めた0.5 mL PBS(-)を加える。(accutaseには酵素反応停止は必要ないとされており、細胞を洗う意味でPBSを添加する。)
・遠心 200Xg 5分間(RT)(ラボのエッペン卓上遠心機では1500rpmぐらい)後、上清を捨てる。遺伝子導入処理をせずに再播種したものをVehicleとした。Cell isolation method (reseeding); the experimental protocol was as follows.
・ Wash 3 times with PBS (-) warmed to 37 ℃.
・ Give 0.5 mL accutase (innovative cell technologies) warmed to 37 ℃ and leave it at 37 ℃ for 30 minutes in a CO 2 incubator.
・ Check that 30-50% of the cells are rounded and floating from the bottom of the dish, and gently tilt the dish to mix, and 90% of the cells will float from the bottom.
Add 0.5 mL PBS (-) warmed to 37 ° C. (Accutase is not required to stop enzyme reaction, and PBS is added to wash the cells.)
・ After centrifuging 200Xg for 5 minutes (RT) (about 1500rpm for a laboratory Eppen table centrifuge), discard the supernatant. Vehicles that had been replated without gene transfer were designated as Vehicles.
Neon(登録商標) Transfection system kit (Invitrogen)を利用したエレクトロポレーションによる手法は下記に示すものであった。
・iPS細胞由来分化心筋細胞を準備する。ここでは、iCell-CM (CDI社)を用いた。
・解凍してラミニンコートしたガラスベースディッシュ(IWAKI 3971-035)に前培養したiCell-CM (CDI社)を前述の方法で単離する。
・アクターゼで単離したiPS心筋細胞をPBSでwash後、遠心(200Xg, 5分間)
・プラスミド溶液を作成
Neon Transfection system kit 100μL(Invitrogen #MPK10025)
Buffer R:100μL Plasmid:1μg
・Neon チューブにBuffer Eを3 mL入れてピペットステーションにセットする。
・細胞の遠心終了後、細胞の上清を100μL程度残してブルーチップで吸い取る。
・イエローチップでしっかりと上清を取り除く。
・プラスミド溶液に細胞を懸濁して、1.5 mLエッペンチューブ(蛋白非吸着タイプ)に移す。
・Neon 100μL tipで細胞懸濁液を吸い上げ、泡を立てずに機器にセットする。
・エレクトロポレーションを行う。 The electroporation method using Neon (registered trademark) Transfection system kit (Invitrogen) was as follows.
Prepare iPS cell-derived differentiated cardiomyocytes. Here, iCell-CM (CDI) was used.
Isolate iCell-CM (CDI) pre-cultured on a glass base dish (IWAKI 3971-035) that has been thawed and laminin-coated by the method described above.
・ After washing iPS cardiomyocytes isolated with actase with PBS, centrifuge (200Xg, 5 minutes)
・ Plasmid solution
Neon Transfection system kit 100μL (Invitrogen # MPK10025)
Buffer R: 100μL Plasmid: 1μg
・ Place 3 mL of Buffer E in a Neon tube and set it in the pipette station.
・ After centrifuging the cells, leave about 100 μL of cell supernatant and blot with a blue chip.
・ Remove the supernatant with a yellow tip.
・ Suspend cells in the plasmid solution and transfer to a 1.5 mL Eppendorf tube (protein non-adsorption type).
・ Draw up the cell suspension with a Neon 100μL tip and set it on the instrument without creating bubbles.
・ Perform electroporation.
Voltage:1650 V、Width:10 ms、Pulse Number:3 (京大プロトコル)
・温めておいたガラスベースディッシュ(IWAKI 3971-035)にすばやく撒き、CO2インキュベーターへ入れて、実験実施まで培養(2日後写真ビデオ撮影)。 *細胞のプラスミド溶液への懸濁から播種までは1サンプルずつ行う。Voltage: 1650 V, Width: 10 ms, Pulse Number: 3 (Kyoto University protocol)
・ Swiftly sprinkle on a warm glass-based dish (IWAKI 3971-035), place in a CO 2 incubator, and incubate until the experiment is conducted (photographed after 2 days). * Perform one sample at a time from cell suspension to seeding.
Lipofectamin2000 (Invitrogen)を利用した手法は下記に示すものであった。即ち、ガラスボトムディッシュに再播種したiCell-CM(1枚ごとの細胞数100-1000個)が定着したのを確認し(2日以上後)、リポフェクタミン2000 (Invitrogen)のプロトコルに従い、以下の順に遺伝子導入を行った。
・50μL Opti-mediumにcDNA 1μg を加え、5分間静置。
・50μL Opti-mediumにLipofectamine2000 2μLを加え、5分間静置。
・上述のそれらの溶液を混合し、20分間静置(室温)。
・準備したiCell-CMに加え、CO2インキュベーターで 37 ℃で培養。
・24時間後に培地交換、さらに24時間後撮影(実験実施)。The method using Lipofectamin2000 (Invitrogen) was as follows. That is, confirm that iCell-CM (100-1000 cells per plate) replated on a glass bottom dish has settled (after 2 days or more), and follow the protocol of Lipofectamine 2000 (Invitrogen) in the following order: Gene transfer was performed.
・ Add 1 μg of cDNA to 50 μL Opti-medium and let stand for 5 minutes.
・ Add 2μL of Lipofectamine2000 to 50μL Opti-medium and let stand for 5 minutes.
-Mix the above solutions and let stand for 20 minutes (room temperature).
・ In addition to the prepared iCell-CM, culture in a CO 2 incubator at 37 ° C.
・ Change the medium after 24 hours, and take pictures after 24 hours (experiment).
蛍光倒立顕微鏡(Olympus IX-71,X40 油浸対物レンズ)にて、GFP蛍光を観察した(キセノンランプ、Ex; 492 ± 9 nm, Em; 530 ± 18 nm)。イメージ撮影と動画撮影は、冷却CCDカメラ(CoolSNAP HQ, Photometrics)で行い、MetaMorphソフトウェア(Molecular Devices 社)でデータ処理および解析を行った。同条件(解像度,シャッター時間等)で取得したGFP画像は全て同条件(カラースケール,閾値等)の処理を施した。 GFP fluorescence was observed with a fluorescence inverted microscope (Olympus IX-71, X40 oil immersion objective lens) (xenon lamp, Ex; 492 ± 9 nm, Em; 530 ± 18 nm). Image shooting and video shooting were performed with a cooled CCD camera (CoolSNAP HQ, Photometrics), and data processing and analysis were performed with MetaMorph software (Molecular Devices). All GFP images acquired under the same conditions (resolution, shutter time, etc.) were processed under the same conditions (color scale, threshold, etc.).
平均動き量は、ビデオ動画からブロックマッチングアルゴリズム(前の画像から動いたベクトル値をブロックごとに計算する古典的手法)を応用したHayakawa T et al., Tissue Eng Part C Methods 18:21-32:2012 の方法に基づいて算出し、動画撮影中(3秒間)の変化を解析した。
17.KCNJ2を過剰発現したiPS心筋シートにおける頻度依存性
iCell-CM(CDI社)は、CDI社のプロトコルとNakamura et al. J Pharmacol Sci 124:494-501 (2014)に従い、凍結バイアルから解凍し、35 mmもしくは60 mmプラスチックディッシュ上で前培養を行った。培地を始め試薬類は全て細胞に同封されるキットのものを使用した。初回解凍後は、バイアル当たり6 cmディッシュ3枚の底面積を基準にして単層培養を行い(前培養)、4日後にMEA用のチャンバー(マルチチャンネルシステム社)に細胞を撒き換えた。The average amount of motion is calculated using a block matching algorithm (a classical method of calculating vector values moved from the previous image for each block) from a video movie. Hayakawa T et al., Tissue Eng Part C Methods 18: 21-32: Calculated based on the 2012 method and analyzed changes during video recording (3 seconds).
17. Frequency dependence in iPS myocardial sheet overexpressing KCNJ2
iCell-CM (CDI) is thawed from frozen vials according to CDI protocol and Nakamura et al. J Pharmacol Sci 124: 494-501 (2014) and pre-cultured on 35 mm or 60 mm plastic dishes. It was. All the reagents including the medium were from the kit enclosed with the cells. After the first thawing, monolayer culture was performed on the basis of the bottom area of three 6 cm dishes per vial (pre-culture), and after 4 days, the cells were changed into a chamber for MEA (Multichannel System).
マルチチャンネルシステム社のMEAシステムを用いて細胞外電位の記録を行った。 Extracellular potentials were recorded using a multi-channel system MEA system.
MEAマルチ電極アレーディッシュの中央にラミニン/poly-D-lysineをコートして、前培養してあったiCell-CMを撒き直す。MEAの電極上に心筋シートを作成するには工夫が必要であり、バイオリサーチセンター社では、電極アレイを中心としてプラスチックの筒を付属することが出来る。その筒に単離した細胞を注入して心筋シートを作成する事が出来た。KCNJ2を遺伝子導入するためのアデノウイルスは、MEAディッシュに撒き直す際に添加した。 Coat laminin / poly-D-lysine in the center of the MEA multi-electrode array dish and re-spread the pre-cultured iCell-CM. In order to create a myocardial sheet on the MEA electrode, it is necessary to devise a device, and BioResearch Center can attach a plastic tube around the electrode array. The myocardial sheet could be made by injecting the isolated cells into the tube. Adenovirus for gene transfer of KCNJ2 was added when re-spreading into the MEA dish.
多電極シート(電極のレイアウト:8 X 8)からの信号は、MEA1060 アンプで増幅し、フィールドポテンシャル(FP)の波形を得た。図8Bの波形に示すように、FPの立ち上がりから基線に戻るまでの時間をフィールドポテンシャル持続時間(FPD)として、FPDの値を評価した。測定温度は全て37℃で行った。測定時の細胞外液はiCell-CM Maintenance medium(CDI社)のままで行い、倒立顕微鏡に取り付けた卓上CO2インキュベーター内にディッシュを静置した。The signal from the multi-electrode sheet (electrode layout: 8 × 8) was amplified by the MEA1060 amplifier to obtain the field potential (FP) waveform. As shown in the waveform of FIG. 8B, the time from the rise of FP to the return to the baseline was defined as the field potential duration (FPD), and the value of FPD was evaluated. All measurement temperatures were 37 ° C. The extracellular fluid at the time of measurement was kept in the iCell-CM Maintenance medium (CDI), and the dish was allowed to stand in a desktop CO 2 incubator attached to an inverted microscope.
刺激入力のための電極は、任意の隣り合う2チャンネルを選択した。それ以外の電極からはFPを計測し、再分極のピークが最も顕著に見られる波形を選択してFPDの値を算出した。FPD値の算出に使用したチャネル(一電極に相当)以外に少なくとも3つのチャネルで同じコントロールのFPD値が得られることも確認した。 Arbitrary adjacent two channels were selected as electrodes for stimulus input. FP was measured from the other electrodes, and the waveform with the most prominent repolarization peak was selected to calculate the FPD value. It was also confirmed that the same control FPD value was obtained with at least three channels other than the channel (corresponding to one electrode) used to calculate the FPD value.
データ取得・データ解析は全てマルチチャンネルシステム社のMEAシステムを使用した。CSVファイルからマイクロソフトのExcel上でデータを解析し、統計はGraphpad社のInstatプログラムを用いて、ANOVAもしくはnon-paired t-testで評価した。
18.結果:hiPS細胞由来心筋細胞の特徴
hiPS細胞由来心筋細胞を得るために、iPS細胞クローンの201B7を胚様体へ分化させた[図2 A]。心筋細胞への分化を証明するため、心筋のマーカーであるα−アクチニン(α−actinin)、コネキシン43(Cx43)、心臓トロポニンT(TnT)の免疫細胞染色を行い、発現を確認した[図2 B,C]。電気生理学的な特徴として、パッチクランプ法による、hiPS細胞由来心筋細胞の活動電位記録から、多様な波形を示すことが明らかとなった。いくつかの活動電位波形は、結節型、心房筋型、心室筋型に類似した波形を示した[図3]。50%再分極時の活動電位持続時間(APD50)と周期長(Cycle Length)をプロットした図では、これまでの報告通り周期長が延びるに従い、APD50が増大する傾向が見られたとともに、APD50値のばらつきも示された[図4]。また、自発的な活動電位を発生していることから、hiPS細胞の分化段階は、幼若であることを示している。この様な、hiPS細胞由来心筋細胞の、成人心室筋細胞と明らかに異なる電気生理学的特徴は、薬剤の毒性を予測する実験系に使用した場合に問題となり得る。最大拡張期電位(MDP)は、成人心室心筋細胞では約−80mVであるのに対して、hiPS細胞由来心筋細胞では約−55mVと陽性方向にシフトしている[図4 B]。最大拡張期電位の大部分はK+イオン濃度に制御されており、すなわち本来ならばK+電流が深く関係しているが、内向き整流K+電流(IK1)を阻害するBa2+イオンへの曝露が最大拡張期電位に対して影響を及ぼさないことから[図4 B]、hiPS細胞由来心筋細胞においては、内向き整流K+電流(IK1)の関与が小さいことを示している。実際、hiPS細胞由来心筋細胞で記録された−150mVにおけるIK1電流密度(pA/pF)は、非常に小さい(−1.5±0.5pA/pF;[図4C])。また、QT間隔延長を引き起こす、hERGチャネル阻害剤E4031の、hiPS由来心筋細胞活動電位における作用を確認すると、低濃度(10,30nM)の曝露の場合は、心室筋活動電位持続時間が延長し、E4031の除去により回復する[図5 A]。一方、段階的にE4031濃度を上げ、100nMまで上昇させた長時間曝露では、除去の後に脱分極不能となり、活動電位が停止する[図5 B]。臨床において100nM/E4031はQT延長が見られる濃度域であり、拍動が止まるという報告はない。これは、本結果のE4031作用濃度が、臨床における作用濃度と異なることを示し、hiPS細胞由来心筋細胞を安全性薬理試験に用いた場合、生体内での作用濃度を読み誤る恐れがある。以上、hiPS細胞由来心筋細胞と、心室筋型ヒト心筋細胞の特徴の違いを図6にまとめる。これらの結果が、K+チャネルKir2.1をコードするヒトKCNJ2遺伝子を、hiPS細胞由来心筋細胞へ導入する動機づけとなった。
19.結果:KCNJ2遺伝子の導入法の検討
hiPS細胞由来心筋細胞へ、アデノウイルスを介したKCNJ2遺伝子の導入を検討した[図10]。まず初めに、胚様体形成前のhiPS細胞にアデノウイルスを感染させたところ、感染ウイルス量が5000MOI濃度で、ごく一部の細胞にのみ、感染を示すGFPの発現を認めた[図11]。次に、1〜1000MOIまでの感染アデノウイルス量において濃度検討を行い、感染させるタイミングを、分化した胚様体の単一細胞化から8日後に設定して、導入効率を検討したところ、導入効率の向上が見られた[図12]。更なる感染条件検討の結果、遺伝子導入のプロトコールとして、胚様体からiPS細胞を単一細胞化した直後に、100MOIの濃度でアデノウイルス感染を開始し、24時間後にウイルスを除き、感染開始から3日目以降の細胞を実験に使用した。発現効率(GFP陽性率)は、約80%であった。
20.結果:KCNJ2の発現は、hiPS細胞由来心筋細胞の自律収縮を停止させる
KCNJ2遺伝子を導入したhiPS細胞由来心筋細胞クラスターについて、免疫細胞染色により心筋細胞マーカーのα−アクチニン、及び遺伝子導入細胞を意味するGFPの発現を確認した[図13 A,B]。GFPタンパク発現遺伝子のみを導入した、ネガティブコントロール細胞クラスターでは、横紋構造の不明瞭さから、心筋への分化の未成熟さが示されたが[図13 A;右下図中の破線内]、GFP融合KCNJ2遺伝子を導入したクラスターでは、過剰発現するKCNJ2(緑)が、α−アクチニン(赤:AAN)の発現を促進し、明瞭な横紋、すなわち成熟した筋節構造の構築が確認できた[図13 B;右下図中の白線内]。KCNJ2遺伝子の導入は、GFPの蛍光シグナルにより確認された[図13 D]。また、201B7細胞株及び253G1細胞株由来の心筋細胞、並びに、iCell心筋細胞、ヒトES細胞由来心筋細胞の全てにおいて、KCNJ2遺伝子の導入により、心筋細胞クラスターの自律拍動性が消失するが、BaCl2への曝露により自律収縮能を回復した[図13 C]。
21.結果:KCNJ2発現及び非発現iCell心筋細胞の細胞外電位記録
iCell心筋細胞シートの、非電気刺激における細胞外電位記録及び、ハイスピードビデオカメラが捉えた収縮する細胞の挙動について検討を行った[図14]。細胞外電場電位の記録には、高純度に精製されたiCell心筋細胞を用い、シート状心筋細胞の電気生理学的特徴を捉える手段として一般的な、MEAシステムを使用した。GFPのみ遺伝子導入された、アデノウイルス感染iCell心筋細胞シート(陰性コントロール)は、細胞外電場電位記録上で周期的な自律性の電気発火を呈した(0.95±0.21Hz,n=5)[図14 A上段]。一方、KCNJ2遺伝子を発現するiCell心筋細胞シートは、完全に静止状態を示した[図14 A中段]。細胞外液中へのBaCl2(0.5mM)投与により内向き整流K+電流(IK1)を阻害すると、細胞外電場電位における自律性の興奮状態が惹起され、自律性の電気発火が開始する[図14 A下段]。従って、自律拍動の抑制には、静止状態のiCell心筋細胞上に過剰発現するKCNJ2遺伝子、つまり、Kir2.1チャネル(IK1電流)の寄与が示唆された。また、KCNJ2を導入した静止状態のiCell心筋細胞シートは、周波数の変換による、電気刺激頻度の増減に依存した拍動性を示した[図14 B,C]。この電気刺激/周波数依存性の拍動は、運動ベクトル解析(Motion vector analysis)により解析を行った。周波数を上げるにつれ、収縮−弛緩持続時間は短縮していったことから、遅延性の整流性K+電流の寄与が示唆された[図14 D]。
22.結果:ヒトiPS細胞由来心筋細胞上に過剰発現させたKCNJ2遺伝子によるKir2.1チャネル(IK1電流)の機能性
Kir2.1チャネルは、KCNJ2遺伝子導入により機能的に過剰発現しており[図15]、GFPの発現は、KCNJ2タンパク発現の有無を確認するための指標として扱われる。Kir2.1チャネルの機能的発現を、パッチクランプ法による電流測定により確認した典型例のトレースを[図15 A]に示す。0.5秒間の電位パルス刺激(コマンドパルス)を、保持電位−80mV、−150mVから−40mVまで、10mV刻みで与えた条件で得られたトレースを、重ね合わせた図を解析した結果、コントロールのiCell心筋細胞では、Kir2.1チャネル選択的阻害剤であるBaCl2(0.2mM)により抑制される電流成分を差し引いた成分(Ba2+−subtracted電流)がほとんど発生しないのに対し、KCNJ2遺伝子を導入したiCell心筋細胞では、Ba2+−subtracted電流、つまりIK1電流の発生が確認できた。刺激電位ごとの最大値をBa2+−subtractedトレースから得てプロットした、IK1電流成分を[図15 B]に示す。(GFPのみ;N=9,KCNJ2;n=9)。Bの一(−80から−40mV)の拡大図を[図15 C]に示す。
23.結果:KCNJ2遺伝子を過剰発現するヒトiPS由来心筋細胞の性質
KCNJ2遺伝子が導入されたiCell心筋の単細胞を用いて、パッチクランプ法により活動電位を記録した。KCNJ2の遺伝子導入が、活動電位波形の形態的特徴を劇的に変化させることが示された[図16 A,B]。[図15]の結果から予測されたように、KCNJ2遺伝子を発現するiCell心筋の単細胞は、静止状態であった[図16 B]。また、KCNJ2遺伝子を発現するiCell心筋の単細胞に対し、細胞内通電を行うと、細胞膜電位の細胞内外の極性が入替わるオーバーシュートを引き起こし、心室筋型の活動電位を誘発する[図16 B]。KCNJ2遺伝子導入により、最大拡張期電位(MDP)、dV/dtmax:0相の最大立ち上がり速度(立ち上がり速度;大きいほど心室筋型に近い)及びAPD40-30/APD80-70率(≦1.5では心房筋様、>1.5では心室筋様の活動電位とみなされる)は、心室筋様に変化することが確認できた[図16 C〜F]。また、パッチクランプ法電流固定モードの記録により、KCNJ2遺伝子発現iCell心筋細胞における、代表的な活動電位トレースを解析すると、IK1内向き整流K+電流を阻害するBa2+イオンの投入により、初回電気刺激以降の通電無しで、自発的な活動電位が誘発され[図17 A上段及び中段]、拡張期第4相に脱分極させる活動電位を自律的に発火させることが示された。KCNJ2遺伝子導入された心筋細胞において、内向き整流K+電流(IK1)を阻害するBa2+イオンへの曝露の有無による電気生理学的性質の変化をまとめたところ、Ba2+イオンへの曝露によりMDPは浅くなり[図17 B]、また一方で、50%再分極時の活動電位持続時間(APD50)は延長を呈した[図17 A下段,C]。
24.結果:KCNJ2発現hiPS由来心筋細胞に対するhERG阻害剤(E4031)の効果
選択的IKr阻害剤のE4031は、KCNJ2遺伝子発現の無いhiPS細胞由来心筋細胞において、拡張期電位を顕著に脱分極へと導いた[図18 A,B]。GFPのみを発現するコントロール細胞では、低濃度E4031曝露により活動電位持続時間(APD)が短縮(A)する細胞と、延長(B)する細胞の両ケースが確認された。KCNJ2発現細胞の最大拡張期電位(MDP)は、累積的なE4031の投与に対して変化を示さず[図18 C,D]、一方で、GFPのみを発現するコントロールの細胞においては、E4031濃度上昇にしたがって、MDPが浅く推移した。また、活動電位持続時間(APD)については、KCNJ2発現細胞では、濃度依存的な延長を示した[図18 C,E]のに対し、コントロール細胞においては、平均の値では濃度依存的な短縮を呈した(n=6)[図18 E]。従って、GFPのみを発現するコントロールの細胞の、E4031による活動電位持続時間延長(APD)を立証することは困難であるが[図18 A,B,D,E]、KCNJ2遺伝子導入した静止状態の心筋細胞を用いた場合は、E4031濃度依存的な、活動電位持続時間の延長を記録することが可能であることが示された[図18 C,D,E]。
25.結果:KCNJ2遺伝子発現により、HEK細胞は内向き整流性K+イオン電流を発生する
心筋細胞の代わりにHEK細胞を用いて、KCNJ2遺伝子の導入を行い、内向き整流K+チャネル電流IK1の電流電圧曲線(B)及び、重ね合わせた電流密度トレース(C)を精査したところ、コントロールベクターによりGFPのみを発現するHEK細胞では、内向き整流K+チャネル電流が発生しないのに対し、KCNJ2遺伝子を導入したHEK細胞では、Ba2+感受性の強い内向き整流K+チャネル電流が発生した[図19 B,C]。
26.結果:iPS由来心筋細胞におけるI(f)チャネルの機能的発現
心臓の洞房結節はペースメーカー電位を発生し、心臓における心拍リズムを決定している。洞房結節のペースメーカー細胞においては、特徴的な緩徐脱分極相が心拍数の調節に大きく寄与しており、この相を形成するペースメーカーチャネルとしてI(f)チャネルが知られている。I(f)チャネルは、主に洞房結節、房室結節とプルキンエ束の伝導系に局在しており、過分極電位で活性化することで、主としてNa+が細胞内に流入する。iPS由来心筋細胞において、I(f)チャネル電流の発生、及び、塩化セシウムによるI(f)チャネル電流の選択的抑制が観察された[図20A〜C]。I(f)チャネルは
、自律神経ホルモンによる心拍数調節に重要な役割を果たすことが知られるが、iPS由来心筋細胞にも発現していることが明らかとなった。しかしながら、選択的I(f)チャネル阻害剤である塩化セシウムによる自律拍動への影響には、各群に有意差は認められなかった[図20D]。よって,iPS由来心筋細胞にはI(f)チャネルが機能的に発現しているものの,自律拍動への影響は小さいことが示された.
27.結果:エピソーマル型ベクター遺伝子導入
エピソーマル型ベクターによるKCNJ2遺伝子の導入に成功した。図21は、遺伝子導入をGFP蛍光シグナルとして評価した結果をまとめた図である。まず、Neonシステムによる遺伝子導入についてであるが、図中のGFP蛍光画像において、vehicleはspot的な自家蛍光のみを示したのに対し(図の上段)、Neonシステムによる処理を施した細胞ではタンパク発現を示す細胞全体の蛍光シグナルを観察することが出来た(図の中段;生存した細胞のほぼ90%)。この処理により、半分以上の細胞が死滅した(浮かんだ)ことから、細胞障害は中程度以上であった。All data acquisition and data analysis was performed using the multi-channel system MEA system. Data were analyzed from Microsoft Excel using CSV files, and statistics were evaluated by ANOVA or non-paired t-test using Graphpad's Instat program.
18. Results: Characteristics of hiPS cell-derived cardiomyocytes In order to obtain hiPS cell-derived cardiomyocytes, the iPS cell clone 201B7 was differentiated into embryoid bodies (FIG. 2A). In order to prove differentiation into cardiomyocytes, myocardial markers α-actinin (α-actinin), connexin 43 (Cx43), and cardiac troponin T (TnT) were immunostained to confirm expression [FIG. B, C]. As an electrophysiological feature, it was revealed from action potential recordings of hiPS cell-derived cardiomyocytes by the patch clamp method that various waveforms were exhibited. Several action potential waveforms showed waveforms similar to the nodular, atrial, and ventricular muscles [FIG. 3]. In the plot of action potential duration (APD50) and cycle length (Cycle Length) at 50% repolarization, APD50 tends to increase as the cycle length increases as reported so far. Also shown was a variation in [Figure 4]. In addition, since spontaneous action potentials are generated, the differentiation stage of hiPS cells is young. Such electrophysiological characteristics of hiPS cell-derived cardiomyocytes that are clearly different from adult ventricular myocytes can be problematic when used in experimental systems that predict drug toxicity. The maximum diastolic potential (MDP) is about −80 mV in adult ventricular cardiomyocytes, whereas it shifts in the positive direction to about −55 mV in hiPS cell-derived cardiomyocytes [FIG. 4B]. Most of the maximal diastolic potential is controlled by the K + ion concentration, that is, K + current is deeply related, but to the Ba 2+ ion that inhibits the inward rectifier K + current (IK1). Since exposure does not affect the maximum diastolic potential [FIG. 4B], it is shown that inward rectifier K + current (IK1) is less involved in hiPS cell-derived cardiomyocytes. In fact, the IK1 current density (pA / pF) at −150 mV recorded with hiPS cell-derived cardiomyocytes is very small (−1.5 ± 0.5 pA / pF; [FIG. 4C]). Moreover, when the effect | action in the hiPS origin cardiomyocyte action potential of the hERG channel inhibitor E4031 causing QT interval extension is confirmed, in the case of low concentration (10, 30 nM) exposure, the ventricular muscle action potential duration is prolonged, It recovers by removing E4031 [FIG. 5A]. On the other hand, long-term exposure in which the E4031 concentration is increased stepwise and increased to 100 nM makes it impossible to depolarize after removal and the action potential stops [FIG. 5B]. In clinical practice, 100 nM / E 4031 is a concentration range where QT prolongation is observed, and there is no report that pulsation stops. This indicates that the E4031 action concentration of this result is different from the action concentration in the clinic, and when the hiPS cell-derived cardiomyocytes are used in the safety pharmacology test, the action concentration in vivo may be misread. The difference in characteristics between the hiPS cell-derived cardiomyocytes and the ventricular myocardial human cardiomyocytes is summarized in FIG. These results motivated the introduction of the human KCNJ2 gene encoding the K + channel Kir2.1 into hiPS cell-derived cardiomyocytes.
19. Result: Examination of introduction method of KCNJ2 gene The introduction of KCNJ2 gene via adenovirus was examined into hiPS cell-derived cardiomyocytes [Fig. 10]. First, when hiPS cells before embryoid body formation were infected with adenovirus, the infection virus amount was 5000 MOI, and only a few cells showed the expression of GFP indicating infection [FIG. 11]. . Next, the concentration of infected adenovirus from 1 to 1000 MOI was examined, and the timing of infection was set 8 days after the differentiation of the differentiated embryoid body into a single cell. [Fig. 12]. As a result of further examination of infection conditions, as a protocol for gene transfer, immediately after iPS cells were made into single cells from embryoid bodies, adenovirus infection was started at a concentration of 100 MOI, virus was removed 24 hours later, and from the start of infection Cells from day 3 onwards were used in the experiment. The expression efficiency (GFP positive rate) was about 80%.
20. Result: Expression of KCNJ2 stops the autonomic contraction of hiPS cell-derived cardiomyocytes. About the hiPS cell-derived cardiomyocyte cluster into which the KCNJ2 gene has been introduced, it means α-actinin, a cardiomyocyte marker, and gene-transferred cells by immunocytostaining The expression of GFP was confirmed [FIGS. 13A, B]. In the negative control cell cluster into which only the GFP protein-expressing gene was introduced, the immaturity of the striated structure showed immature differentiation into myocardium [FIG. 13A; in the broken line in the lower right figure] In the cluster into which the GFP-fused KCNJ2 gene was introduced, overexpressed KCNJ2 (green) promoted the expression of α-actinin (red: AAN), and it was confirmed that a clear striated, ie, a mature sarcomere structure was constructed. [FIG. 13B: In the white line in the lower right figure]. The introduction of the KCNJ2 gene was confirmed by the fluorescence signal of GFP [FIG. 13D]. In addition, in all the cardiomyocytes derived from 201B7 cell line and 253G1 cell line, as well as iCell cardiomyocytes and human ES cell-derived cardiomyocytes, the autonomous pulsation of the cardiomyocyte cluster is lost by the introduction of the KCNJ2 gene. Exposure to 2 restored autonomic contractility [FIG. 13C].
21. Results: Extracellular potential recording of iCell cardiomyocytes expressed and not expressed with KCNJ2 We investigated the extracellular potential recording of non-electrical stimulation of iCell cardiomyocyte sheets and the behavior of contracting cells captured by a high-speed video camera. 14]. The extracellular electric field potential was recorded using iCell cardiomyocytes purified to a high purity, and a general MEA system was used as a means for capturing the electrophysiological characteristics of sheet-like cardiomyocytes. The adenovirus-infected iCell cardiomyocyte sheet (negative control) into which only GFP was introduced exhibited periodic autonomous electrical firing on the extracellular electric field potential recording (0.95 ± 0.21 Hz, n = 5). ) [FIG. 14A top]. On the other hand, the iCell cardiomyocyte sheet expressing the KCNJ2 gene was completely quiescent [FIG. 14A middle]. Inhibition of inward rectification K + current (IK1) by administration of BaCl 2 (0.5 mM) into the extracellular fluid causes an autonomous excitement state at the extracellular electric field potential and initiates autonomous electrical firing. [FIG. 14A Lower]. Therefore, it was suggested that KCNJ2 gene overexpressed on quiescent iCell cardiomyocytes, that is, Kir2.1 channel (IK1 current) contributes to suppression of autonomous pulsation. In addition, the stationary iCell cardiomyocyte sheet into which KCNJ2 was introduced showed pulsation depending on the increase / decrease in electrical stimulation frequency due to frequency conversion [FIGS. 14B, C]. This electrical stimulation / frequency-dependent pulsation was analyzed by Motion vector analysis. As the frequency was increased, the contraction-relaxation duration decreased, suggesting the contribution of delayed rectifying K + current [FIG. 14D].
22. Results: Functionality of Kir2.1 channel (IK1 current) by KCNJ2 gene overexpressed on human iPS cell-derived cardiomyocytes Kir2.1 channel is functionally overexpressed by introduction of KCNJ2 gene [FIG. 15] The expression of GFP is treated as an index for confirming the presence or absence of KCNJ2 protein expression. A typical trace in which the functional expression of the Kir2.1 channel was confirmed by current measurement by the patch clamp method is shown in FIG. 15A. As a result of analyzing the superimposed diagram of the traces obtained by applying a 0.5 second potential pulse stimulus (command pulse) from the holding potential of −80 mV, from −150 mV to −40 mV in 10 mV increments, In iCell cardiomyocytes, a component (Ba 2+ -subtracted current) obtained by subtracting the current component suppressed by the Kir2.1 channel selective inhibitor BaCl 2 (0.2 mM) is hardly generated, whereas the KCNJ2 gene is In the introduced iCell cardiomyocytes, generation of Ba 2+ -subtracted current, that is, IK1 current was confirmed. The IK1 current component plotted from the maximum value for each stimulation potential obtained from the Ba2 + -subtracted trace is shown in FIG. 15B. (GFP only; N = 9, KCNJ2; n = 9). An enlarged view of one of B (-80 to -40 mV) is shown in FIG. 15C.
23. Results: Properties of human iPS-derived cardiomyocytes overexpressing the KCNJ2 gene Action potentials were recorded by the patch clamp method using single cells of iCell myocardium into which the KCNJ2 gene was introduced. It has been shown that gene transfer of KCNJ2 dramatically changes the morphological characteristics of the action potential waveform [FIGS. 16A, B]. As predicted from the results in FIG. 15, iCell myocardial single cells expressing the KCNJ2 gene were in a quiescent state (FIG. 16B). In addition, when an intracellular current is applied to a single cell of an iCell myocardium expressing the KCNJ2 gene, an overshoot in which the polarity of the cell membrane potential is switched inside and outside the cell is induced to induce a ventricular muscular action potential [FIG. 16B]. . By introducing the KCNJ2 gene, the maximum diastolic potential (MDP), dV / dtmax: the maximum rising speed of the zero phase (rising speed; the larger it is, the closer to the ventricular muscle type) and the APD 40-30 / APD 80-70 rate (≦ 1. It was confirmed that the action potential of atrial muscle-like in 5 was regarded as a ventricular-like action potential in> 1.5 [FIGS. 16C to F]. In addition, when a typical action potential trace in a KCNJ2 gene-expressing iCell cardiomyocyte is analyzed by recording in a patch clamp current fixing mode, the initial electrical stimulation is performed by introducing Ba 2+ ions that inhibit IK1 inward rectification K + current. Without subsequent energization, a spontaneous action potential was induced [FIG. 17A, upper and middle stages], indicating that the action potential that depolarizes into the diastolic phase 4 is autonomously ignited. In KCNJ2 transgenic cardiomyocytes, it was summarized changes in electrophysiological properties with and without exposure to Ba 2+ ions that inhibit the inward rectifier K + current (IK1), MDP exposure to Ba 2+ ions On the other hand, the action potential duration (APD50) at 50% repolarization was prolonged [FIG. 17A, bottom, C].
24. Results: Effect of hERG inhibitor (E4031) on KCNJ2-expressing hiPS-derived cardiomyocytes The selective IKr inhibitor E4031 significantly led to diastolic potential depolarization in hiPS cell-derived cardiomyocytes without KCNJ2 gene expression [FIG. 18 A, B]. In the control cells expressing only GFP, both cases of action potential duration (APD) shortening (A) and extension (B) cells by low concentration E4031 exposure were confirmed. The maximal diastolic potential (MDP) of KCNJ2-expressing cells did not change with cumulative E4031 administration [FIGS. 18C, D], whereas in control cells expressing only GFP, the E4031 concentration As the rise, MDP remained shallow. In addition, the action potential duration (APD) showed a concentration-dependent extension in the KCNJ2-expressing cells [FIG. 18C, E], whereas in the control cells, the average value showed a concentration-dependent shortening. (N = 6) [FIG. 18E]. Therefore, although it is difficult to prove action potential duration extension (APD) by E4031 in a control cell expressing only GFP [FIGS. 18A, B, D, E], a quiescent state in which the KCNJ2 gene was introduced. When cardiomyocytes were used, it was shown that it was possible to record an extension of action potential duration dependent on E4031 concentration [FIGS. 18C, D, E].
25. Results: HEK cells generate inward rectifying K + ion currents due to KCNJ2 gene expression. HEK cells are used instead of cardiomyocytes to introduce the KCNJ2 gene, and the inward rectifying K + channel current IK1 current voltage. Examination of the curve (B) and the superimposed current density trace (C) revealed that inwardly rectified K + channel current was not generated in HEK cells expressing only GFP by the control vector, whereas the KCNJ2 gene was introduced. HEK cells generated inward rectifier K + channel currents with strong Ba 2+ sensitivity [FIGS. 19B, C].
26. Results: Functional expression of I (f) channels in iPS-derived cardiomyocytes The sinoatrial node of the heart generates a pacemaker potential and determines the heart rhythm in the heart. In the pacemaker cells of the sinoatrial node, the characteristic slow depolarization phase greatly contributes to the regulation of the heart rate, and the I (f) channel is known as a pacemaker channel that forms this phase. The I (f) channel is mainly localized in the conduction system of the sinoatrial node, the atrioventricular node and the Purkinje bundle, and is activated by a hyperpolarizing potential, whereby Na + mainly flows into the cell. In iPS-derived cardiomyocytes, generation of I (f) channel current and selective suppression of I (f) channel current by cesium chloride were observed [FIGS. 20A to 20C]. The I (f) channel is known to play an important role in the regulation of heart rate by autonomic neurohormones, but it has been revealed that it is also expressed in iPS-derived cardiomyocytes. However, there was no significant difference in the effect of cesium chloride, a selective I (f) channel inhibitor, on autonomic pulsation in each group [FIG. 20D]. Thus, although the I (f) channel is functionally expressed in iPS-derived cardiomyocytes, it has been shown that the influence on the autonomic pulsation is small.
27. Result: Episomal vector gene introduction The KCNJ2 gene was successfully introduced using an episomal vector. FIG. 21 is a table summarizing the results of evaluating gene transfer as a GFP fluorescence signal. First, regarding the gene transfer using the Neon system, the vehicle showed only spot-like autofluorescence in the GFP fluorescence image in the figure (upper part of the figure), whereas the cells treated with the Neon system showed protein. The fluorescence signal of the whole cell showing expression could be observed (middle of the figure; almost 90% of the surviving cells). With this treatment, more than half of the cells were killed (floated), so cell damage was moderate or better.
次に、Lipofectamine2000を用いた遺伝子導入についてであるが、図の最下段に示すように細胞クラスターの一部の細胞で強い蛍光シグナルを観察することが出来た。図の最下段のGFP蛍光画像では、上部の細胞には遺伝子導入されているが、下部の細胞は自家蛍光のみであり、遺伝子が導入されなかったことを示している。全体としては,10%以下の導入効率であったが、細胞障害は弱く、死滅した細胞もクラスターの縁に少し観察されるのみであった。 Next, regarding gene transfer using Lipofectamine 2000, a strong fluorescent signal could be observed in some cells of the cell cluster as shown in the bottom of the figure. The GFP fluorescence image at the bottom of the figure shows that the gene was introduced into the upper cell, but the lower cell was only autofluorescent, indicating that no gene was introduced. Overall, the introduction efficiency was 10% or less, but the cell damage was weak, and only a few dead cells were observed at the edge of the cluster.
アデノウイルスベクターを用いてKCNJ2を遺伝子導入したときと同様に、KCNJ2遺伝子導入により、拍動心筋の動きが停止した。 In the same manner as when KCNJ2 was introduced using an adenovirus vector, the pulsating myocardial movement was stopped by introduction of the KCNJ2 gene.
図22は、図21のうち、vehicle(上段)とLipofectamine2000(下段)のビデオ画像(3秒間)から算出した平均動きベクトル量の時間変化を示している。図に示すように、KCNJ2-EGFPの遺伝子導入により、細胞の動きが完全に停止した。一方で、vehicle細胞は拍動しており、動きベクトルの活発な変化が見られた。以上の実験結果は、エピソーマル型ベクターによるKCNJ2-EGFP遺伝子導入によっても、本発明の機能変化をもたらすことが可能であることを示した。
28.結果:KCNJ2を過剰発現したiPS心筋シートから計測したFPDの頻度依存性の検討
図8にKCNJ2を過剰発現したiCell-CMシートから計測したFPD(フィールドポテンシャル持続時間)の頻度依存性を示す。刺激頻度は0.5 Hzから2 Hzである。図8Aには、FP波形の典型例を示す。図8B上段には、異なる頻度(0.5, 1, 2 Hz)によるFP波形を重ね合わせた図を示している(一つの心筋シート標本から得られたデータ)。図8B下段には、7例から得られたFPD値の頻度依存性の結果をまとめている。黒いシンボルは個々のシートの値であり、異なる頻度の値を点線で結び、頻度依存性を示している。白抜きシンボルで平均値を示しており、頻度(Hz)の値が増えるほどFPDが減るという逆相関が見られた。この結果は、QT間隔や活動電位持続時間に見られる頻度依存性(逆頻度依存とも言う)を良く反映する結果であり、当該発明による処理を行う事ではじめて観察することに成功した。FIG. 22 shows temporal changes in the average motion vector amount calculated from the video images (3 seconds) of vehicle (upper) and Lipofectamine 2000 (lower) in FIG. As shown in the figure, the cell movement was completely stopped by the gene introduction of KCNJ2-EGFP. On the other hand, vehicle cells were pulsating, and a vigorous change in the motion vector was observed. The above experimental results showed that the functional change of the present invention can also be brought about by introduction of the KCNJ2-EGFP gene with an episomal vector.
28. Results: Examination of frequency dependence of FPD measured from iPS myocardial sheet overexpressing KCNJ2 FIG. 8 shows frequency dependence of FPD (field potential duration) measured from iCell-CM sheet overexpressing KCNJ2. The stimulation frequency is 0.5 Hz to 2 Hz. FIG. 8A shows a typical example of an FP waveform. The upper part of FIG. 8B shows a diagram in which FP waveforms with different frequencies (0.5, 1, 2 Hz) are superimposed (data obtained from one myocardial sheet specimen). The lower part of FIG. 8B summarizes the frequency dependence results of the FPD values obtained from 7 cases. Black symbols are values of individual sheets, and values of different frequencies are connected by dotted lines to indicate frequency dependency. The average value is indicated by white symbols, and an inverse correlation was observed in which the FPD decreased as the frequency (Hz) increased. This result is a result that well reflects the frequency dependency (also referred to as inverse frequency dependency) seen in the QT interval and action potential duration, and was successfully observed for the first time by performing the processing according to the present invention.
心電図のQT間隔、ランゲンドルフ心臓標本のQT間隔もしくはMAP、心筋細胞標本の活動電位幅(APD)には頻度依存性があることがよく知られており、その性質は不整脈発生において重要であることが知られている(Viswanathan PC et al., Circulation 99:2466-2474:1999)。自律拍動をする未処理のiPS心筋(平均拍動能 約1 Hz)には低頻度刺激(0.5 Hzなど)を入力することは不可能であるため、刺激頻度依存性を評価することは出来ないので、当該発明による処理をiPS心筋に施したところ、刺激頻度0.5 Hzから5 Hzまでの刺激印加に追随して心筋シートを拍動させることに成功した。2Hz以上に刺激頻度を上げていくとビートごとのFPDが安定しない現象が見られ、5Hzまでの刺激印加に対してはおおむね追随するという結果が得られた。 It is well known that ECG QT interval, Langendorff heart QT interval or MAP, and cardiomyocyte action potential width (APD) are frequency-dependent, and their properties may be important in arrhythmia development. (Viswanathan PC et al., Circulation 99: 2466-2474: 1999). Since it is impossible to input low-frequency stimuli (such as 0.5 Hz) to unprocessed iPS myocardium (with an average pulsatile capacity of about 1 Hz) that does autonomous pulsation, it is not possible to evaluate the stimulus frequency dependence Therefore, when the treatment according to the present invention was applied to the iPS myocardium, the myocardial sheet was successfully beaten following the application of the stimulus with a stimulation frequency of 0.5 Hz to 5 Hz. When the frequency of stimulation was increased to 2 Hz or more, the phenomenon that the FPD for each beat was not stable was observed, and the result was that it generally followed the stimulus application up to 5 Hz.
代表的QT延長薬剤であるE4031存在下で同様の実験を行ったところ、2 HzではビートごとのFPDの不安定性が上がり、5Hz刺激には全く追随せず、スパイラルリエントリーを示唆するFP波形が観察された。よって、10 nM E4031において、催不整脈性を示す2つのパラメーター;ビートごとのFPD値のばらつきおよび高頻度における刺激不追随性、が顕著に上がることが示された。 A similar experiment was conducted in the presence of E4031, a typical QT prolonging drug. At 2 Hz, the FPD instability increased at every beat, and the FP waveform suggesting spiral reentry did not follow the 5 Hz stimulus at all. Observed. Therefore, in 10 nM E4031, it was shown that two parameters indicating arrhythmogenicity: variation in FPD value from beat to beat and stimulus follow-up at high frequency significantly increase.
即ち、図9は、KCNJ2を過剰発現したiCell-CMシートから高頻度刺激により計測したFP波形に対するQT延長薬E4031の影響を示す図である。刺激頻度2 Hz(上段)および5 Hz(下段)の結果を示す。左側にはコントロール(薬物投与前)、右側にはE4031(10 nM 5分間添加)存在下のFP波形を示す。 That is, FIG. 9 is a figure which shows the influence of the QT prolongation drug E4031 with respect to the FP waveform measured by the high frequency stimulation from the iCell-CM sheet which overexpressed KCNJ2. The results of stimulation frequency 2 Hz (upper) and 5 Hz (lower) are shown. The left side shows the FP waveform in the presence of control (before drug administration) and the right side in the presence of E4031 (added at 10 nM for 5 minutes).
まず2 Hzの結果であるが、コントロールでは刺激後の再分極ピークまでのFPD値は150-200 msの範囲内でのばらつきであったが、E4031添加後には150-350 msとFPD値ばらつきの程度が広がった。5Hzの結果では、コントロールの時には図中の矢印は刺激から、大体180 ms後に再分極が見られることが多かった(7回中5回)。一方で、E4031添加後には刺激後一定の時間に再分極することはなく、ランダムに再分極のピークが見られたことから(下段右:矢印参照)リエントリーが発生している特徴を示すFP波形が得られた.
29.まとめ:図23に示すように、hiPS由来心筋細胞は、自律拍動する幼若な細胞であり、E4031によるhERGチャネル阻害が、最大拡張期電位(MDP)を低下させるため、活動電位持続時間(APD)の延長を安定的に計測することができない。一方、KCNJ2遺伝子を導入することで成熟化したhiPS由来心筋細胞は、活動電位の発火が電気刺激依存的になり、かつ、E4031によるhERGチャネル阻害が、活動電位持続時間(QT間隔)を延長させており、より臨床への外挿性に優れた心筋モデル細胞であると言える。一般にQT延長薬の薬物評価の解析では、フィールドポテンシャル持続時間(FPD)という指標が良く用いられているが、FPDの始点は、Naチャネルでのイオン流入による、脱分極の大きなピークとして同定される一方で、FPDの終点は、ピークもピークの終わりもはっきりしないばかりか、研究者により測り方の見解が異なるため、データ解析を困難にしている。図24に示すように、MEAシステム(Microelectrode Array/マルチ電極アレイ)による細胞外電位の測定結果では、GFPのみを発現するコントロール細胞では、FPDのピーク及びピークの終点が明瞭では無いが(A上段)、KCNJ2遺伝子を導入された細胞では、カルシウムチャネルによる再分極が急峻になるため、顕著な2ndピークが出現し(A下段)、はじめのピークと2ndピーク間をFPDとすれば、解析誤差を回避することができる。現行では、A上段に示す様なFPD1やFPD2を測定に用いており、従って、ひとつの測定結果に対して異なる結論が導かれる[FPD1の方法で計測している例:Tanaka T, et al. BBRC 2009;385:497-502. FPD2の方法で計測している例:Matsa E et al., Eur Heart J 2011;32:952-962]。また、GFPのみを発現するコントロール細胞では、10nMのE4031作用に対して、心拍が落ち、FPD1、FPD2共に延長を呈するものの、ピーク及びピークが基線に戻るポイントの決定が困難であるのに対し(B)、KCNJ2遺伝子導入細胞では、同濃度のE4031作用下でも、FPDの2ndピークが顕著である(C)。(B)の様な、現行で用いられている細胞では、FPD延長の程度が各々の拍によって異なり、正確な判定に影響を及ぼすが、KCNJ2遺伝子を導入した細胞を用いれば、薬物による再分極の影響を正確に読み取ることができる。更に、KCNJ2導入細胞の顕著な2ndピークの特徴を生かせば、比較的単純なプログラムでピーク検出ができるため、ハイスループットスクリーニングといった高精度高効率解析システムが、現実的に開発可能となる。First, the 2 Hz results show that in the control, the FPD value until the repolarization peak after stimulation was within the range of 150-200 ms, but after the addition of E4031, the FPD value variation was 150-350 ms. The degree spread. In the result of 5 Hz, the arrow in the figure at the time of control often showed repolarization approximately 180 ms after stimulation (5 times out of 7). On the other hand, after addition of E4031, there was no repolarization at a fixed time after stimulation, and a repolarization peak was seen at random (bottom right: see arrow). A waveform was obtained.
29. Summary: As shown in FIG. 23, hiPS-derived cardiomyocytes are young cells that autonomously beat, and hERG channel inhibition by E4031 reduces the maximum diastolic potential (MDP). The extension of APD) cannot be measured stably. On the other hand, in hiPS-derived cardiomyocytes matured by introducing the KCNJ2 gene, action potential firing is dependent on electrical stimulation, and hERG channel inhibition by E4031 prolongs the action potential duration (QT interval). Therefore, it can be said that it is a myocardial model cell with excellent clinical extrapolation. In general, in the analysis of drug evaluation of QT prolonging drugs, an index called field potential duration (FPD) is often used, but the starting point of FPD is identified as a large peak of depolarization due to ion inflow in Na channel. On the other hand, the end point of FPD not only makes the peak and the end of the peak unclear, but also makes the data analysis difficult because the researcher has different views on how to measure. As shown in FIG. 24, in the measurement result of the extracellular potential by the MEA system (Microelectrode Array / multi-electrode array), in the control cell expressing only GFP, the peak of FPD and the end point of the peak are not clear (A upper row). ) In cells into which the KCNJ2 gene has been introduced, repolarization by the calcium channel becomes steep, so that a remarkable 2nd peak appears (lower A). If FPD is used between the first peak and the 2nd peak, an analysis error will occur. It can be avoided. At present, FPD1 and FPD2 as shown in the upper part of A are used for the measurement, and therefore different conclusions are derived for one measurement result [example of measurement using the FPD1 method: Tanaka T, et al. BBRC 2009; 385: 497-502. An example of measurement by the FPD2 method: Matsa E et al., Eur Heart J 2011; 32: 952-962]. In addition, in control cells expressing only GFP, although the heart rate falls and both FPD1 and FPD2 exhibit an extension to 10 nM E4031 action, it is difficult to determine the peak and the point at which the peak returns to the baseline ( B) In the KCNJ2 gene-introduced cells, the 2nd peak of FPD is prominent even under the action of E4031 at the same concentration (C). In currently used cells such as (B), the degree of FPD prolongation varies depending on each beat and affects accurate determination. However, if cells into which the KCNJ2 gene is introduced are used, repolarization by drugs Can be read accurately. Furthermore, since the peak can be detected with a relatively simple program if the remarkable 2nd peak feature of the KCNJ2-introduced cell is utilized, a highly accurate and efficient analysis system such as high-throughput screening can be practically developed.
本発明によると、in vitroにおいて評価の困難であった、被検物質に起因する活動電位持続時間の延長作用を、高い精度で検出することが可能となり、外挿性の高い解析結果が期待できることから、スクリーニング用モデル細胞として、創薬技術への応用に有用である。 According to the present invention, it becomes possible to detect the action potential duration-prolonging effect caused by the test substance, which has been difficult to evaluate in vitro, with high accuracy, and an analysis result with high extrapolation can be expected. Therefore, it is useful for application to drug discovery technology as a model cell for screening.
Claims (10)
1)人工多能性幹細胞(iPS細胞)から心筋細胞又は心筋前駆細胞に分化させる工程(a);
2)工程(a)により得られた心筋細胞又は心筋前駆細胞を含む胚様体若しくはコロニーを、単一細胞に分離する工程(b);
3)KCNJ2遺伝子を組み込み、Kir2.1チャネルを発現可能なベクターを調製する工程(c);
4)工程(b)により分離した細胞を、分離直後から1時間以内に、工程(c)で調製したウイルスベクターに感染させる工程(d);
5)心臓トロポニンT(TnT)、コネキシン43(Cx43)、又はα−アクチニン(α−actinin)のうち少なくとも1つの細胞内因性遺伝子を発現し、かつ、Kir2.1チャネルを発現する細胞を選択する工程(e)A method for producing a myocardial model cell comprising the following steps (a) to (e):
1) a step of differentiation from induced pluripotent stem cells (iPS cells) into cardiomyocytes or myocardial progenitor cells (a);
2) A step (b) of separating the embryoid body or colony containing cardiomyocytes or myocardial progenitor cells obtained in step (a) into single cells;
3) A step of preparing a vector capable of expressing the Kir2.1 channel by incorporating the KCNJ2 gene (c);
4) A step (d) of infecting the cells separated in step (b) with the virus vector prepared in step (c) within 1 hour immediately after the separation;
5) Select a cell that expresses at least one cellular endogenous gene of cardiac troponin T (TnT), connexin 43 (Cx43), or α-actinin and expresses the Kir2.1 channel Step (e)
1)請求項1〜5のいずれかに記載の心筋モデル細胞と被検物質とを接触させる工程(A);
2)電気生理学的試験手法を用いて、被検物質に起因する心筋細胞に対する毒性作用及び/又は変調作用を検出する工程(B);
3)工程(B)の検出結果に基づき、被検物質の心筋細胞に対する毒性作用及び/又は変調作用の有無を判定する工程(C); A screening method for a substance having a toxic effect and / or a modulating effect on cardiomyocytes, comprising the following steps (A) to (C):
1) A step (A) of contacting the myocardial model cell according to any one of claims 1 to 5 with a test substance;
2) A step (B) of detecting a toxic effect and / or a modulation effect on cardiomyocytes caused by a test substance using an electrophysiological test method;
3) A step (C) of determining the presence or absence of a toxic effect and / or a modulation effect on the cardiomyocytes of the test substance based on the detection result of the step (B);
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