著者らは、この出版物の生産コストをカバーする彼らの種類の財政的貢献のためにケアンリサーチ株式会社を認めたいと思います。さらに、イネス・ミューラー氏とステファニー・ケステル氏の優れた技術サポートに感謝します。
著者らの研究は、ドイツ心臓血管研究センター(DZHK)、ドイツフォルシュングスゲミンシャフト(DFG、 ドイツ研究財団、VO 1568/3-1、IRTG1816 RP12、SFB1002 TPA13、ドイツのエクセレンス戦略の下で - EXC 2067/1- 390729940)とエルゼ・クレナー・フレゼニウス・スティフトゥン(EKFS)2016_A20 FS)。

1. 細胞製剤
注:このプロトコルで使用されるヒトiPSCは、健康なドナーから誘導され、前述の12、13、14のように、WNTシグナルおよび乳酸精製技術の完全に定義された小分子変調を使用して単層で区別された。iPSC-CMは、以下に概説する培養培地で2〜3日ごとに維持された。
<ol><li>基礎培地(RPMI 1640)および2%サプリメント(B27)の培地を調製する。4 °Cで保管。 室温(RT)で使用してください。</li>
	<li>基底培地(RPMI 1640)、2%サプリメント(B27)および1:2000 ROCK阻害剤のめっき培地を調製します。4 °Cで保管。 RT で使用します。</li>
	<li>コートは10 mmの丸いガラス#0のカバースリップを1:60の1:60の気に入った基膜マトリックスの150のmmと4の4時間の4°Cでインキュベートする。 注:カバースリップの体積の最適化は、こぼれを防ぐために十分な表面張力を維持しながら、ガラス全体が覆われていることを確認するために必要です。150 μL は、10 mm の丸いカバーリップに推奨されます。バッチサイズ、カバースリップタイプ、カバースリップ容積および培養プレートタイプは、実験者のニーズに適することができる。</li>
	<li>EDTAベースの細胞解離試薬とのiPSC-CM解離を開始します。1,000 μL ピペットで静かに洗い流して、単層が完全に切り離されていることを確認します。</li>
	<li>細胞懸濁液を15mLチューブに移し、二重容積メッキ媒体を加えます。遠心分離機 10分 100 x g.</li>
	<li>ペレットを、メッキ媒体の所望の容積(再懸濁液量)で再懸濁する。手動または電子的にセルをカウントします。</li>
	<li>カバースリップ当たりの最適密度(15,000)を選択し、後で単離された細胞分析を可能にします。</li>
	<li>この望ましい密度ですべてのカバーリップをプレートするために必要な「アクティブ」細胞懸濁液(A)の体積を計算します。次の式を適用し、別のチューブに引き出します。 <img alt="Equation 1" src="/files/ftp_upload/61890/61890equ01v2.jpg">
</li>
	<li>各カバースリップを所望のボリュームに収めるために必要な追加めっき媒体(B)の体積を計算します。次の式を適用し、結果のボリュームをアクティブサスペンションのチューブに追加します。 <img alt="Equation 2" src="/files/ftp_upload/61890/61890equ02v2.jpg">
</li>
	<li>カバースリップからマトリックスを取り外し、セルサスペンション(A+B)の「カバースリップボリューム」を各カバースリップに適用します。細胞の分布を確保するためにチューブ内で定期的に再中断します。</li>
	<li>37°Cで1時間インキュベートする。めっき培地でウェルを軽く充填します。</li>
	<li>24時間後、培地を通常の培養培地と交換し、2~3日毎に維持する。</li>
</ol>2. 実験的なセットアップ
<ol><li>反転した蛍光顕微鏡に40倍の拡大率、高い開口レンズ(N.A:>0.75)を装備して実験を行います。</li>
	<li>高速切り替え暖かい白色LEDを顕微鏡の送信された照明ポートに結合する。透過光路にシンプルな赤色660 nmフィルタを挿入します。 注:この光は、緑色の蛍光信号を汚染することなくサンプルを観察するために、光測定実験を通して活性化することができます。</li>
	<li>フォトメトリー記録用の高速スイッチング470 nm LEDヘッドを取り付けます。470/40励起フィルターを顕微鏡の蛍光ポートに挿入して、LEDによって発生する光をクリーンアップします。 メモ:最適な信号定量を行うため、光出力の高速フィードバック制御を備えた照明システムを推奨します。</li>
	<li>顕微鏡内の鏡ユニットカルーセルに495nmのロングパスビームスプリッターを含む顕微鏡キューブを挿入します。</li>
	<li>可変フィールドダイヤフラムを含む検出アームを顕微鏡Cマウントポートに装着し、対象領域を選択できるようにします。</li>
	<li>別に顕微鏡に光増倍検出器(PMT)とUSBカメラを組み合わせる。これは、排出ガス検出システムの基礎を形成します。</li>
	<li>565 nmのロングパスビームスプリッターと535/50のエミッションフィルタを含むフィルタキューブをPMTポートに挿入します。これは2つの探知器間の放出の光を割る。 注:発光検出システムの送信ポートに取り付けられたカメラは、すべての実験を通して明視野下で透過光を検出することができます。</li>
	<li>PMT を電源装置と PMT アンプに結合します。PMTアンプ出力をデータ収集システムのアナログ入力ピンに接続します。</li>
	<li>PMTからのアナログデータを1kHz以上でフィルタリングします。</li>
	<li>ナイキスト基準を満たし、エイリアスを防ぐために、アナログ信号(2 kHz以上)に存在する最も高い周波数の2倍以上の周波数でデータをデジタル化します。</li>
</ol>VF2.1Cl によるセルラーローディング
注:この染料を含むすべてのステップは、低照度条件で行う必要があります。
<ol><li>タイロードの浴液(mM)を準備する:140 NaCl、10 HEPES、10グルコース、4KCl、1 MgCl2、2CaCl2、pH= 7.35、37°Cに暖かい。</li>
	<li>1,000x VF2.1Clの5 μLと20%可溶化ポロクサマー溶液の50 μLを混合して、マイクロ遠心チューブに溶液をローディングするアリコートを用意します。</li>
	<li>20 mm ペトリ皿に 5 μL の加温タイローデ溶液(合計0.1x染料濃度)を5 mL塗布します。 注意: 最終染料濃度は0.1倍です。これはメーカーが提案した1/10thです 。これにより、リソースを節約し、ごくわずかな細胞毒性を保証し、重要なことに、高い信号対雑音比を持つロードされた細胞からの明確な光信号を保持します。</li>
	<li>iPSC-CMカバースリップを1枚皿に加え、37°Cで20分間インキュベートします。</li>
	<li>加熱された生細胞イメージングチャンバーを組み立てます。顕微鏡の段階に取付け、500 μL の新鮮なタイローデの溶液で満たします。</li>
	<li>37 °Cで新鮮なタイロードの溶液でカバースリップを洗います。</li>
	<li>iPSC-CMカバースリップを、細かいポイント鉗子を使用して、あらかじめ温めた浴室に慎重に塗布してください。 注:チャンバーとその内容物が常に生理学的温度で加熱されていることを確認してください。必要に応じて、温めたタイローデの溶液の連続灌流から始めます。</li>
</ol>4. 電界刺激
注: iPSC-CM の外部トリガーはオプションですが、セルラーダイナミクスと実験パラメータの標準化に役立ちます。分析の容易さを高め、周波数依存効果の調査を可能にします。
<ol><li>5 mm離れた場所に2つの白金電極を付けて、刺激インサートを記録チャンバーに取り付けます。</li>
	<li>外部刺激装置を刺激インサートに接続します。0.5 Hz の 5 ms バイポーラ フィールド パルスに設定します。</li>
	<li>1 Vから上向きの刺激を増加させることにより、最適な刺激電圧を決定します。閾値刺激は、セルが収縮し始める最も低い電圧として定義されます。このしきい値を約25%上回る電圧を適用します。通常の範囲は1 V~30Vです。</li>
	<li>外部刺激器で刺激周波数を固定するか、取得ソフトウェアでトリガーします。</li>
</ol>5. 光学的なアクションポテンシャル獲得
注: このプロトコルは、取得および分析に商用ソフトウェアを使用します。
<ol><li>透過光路とUSBカメラを使用して、明視野ビューで筋細胞を可視化します。</li>
	<li>孤立した細胞を選択し、対象の細胞からの光のみを監視することを保証するフィールドダイヤフラムで光学路をしっかりと切り取ります。</li>
	<li>PMTアンプを起動し、PMT電源を750Vに設定します。</li>
	<li>刺激プロトコル(ステップ4を参照)を取得ソフトウェアと一緒に実行し、同時に470 nm励起光をアクティブにします。後者はリモートパネルを介して行うか、固定強度(TTL信号)で自動化することができます。</li>
	<li>PMTアンプのゲインとオフセットを調整して、信号が飽和せず、記録システムの検出範囲に最適化されていることを確認します。</li>
	<li>安定したアクション電位を確実に検出する、10のスイープを記録します。</li>
	<li>記録を続け、すぐに顕微鏡ステージを動かして、細胞を欠いた領域からバックグラウンド信号を短時間取得します。励起ライトをオフにします。 注: この背景値 (Fオフセット ) は、任意のバックグラウンド蛍光を考慮するために使用されます。</li>
	<li>所望ならば、1μMニフェジピンなどの参照薬を局所的にパーフューズし、薬理学的操作に対する細胞応答を同定する。</li>
	<li>順次的に、新しいセルを選択するたびに、ステップ 5.2 ~ 5.7 を繰り返します。必要に応じてカバーリップを交換して、1座って高い実験回転率を確保します。 注 : 読み込みおよびイメージ取得のプロトコルについては 、図 2を参照してください。</li>
</ol>6. データ分析
<ol><li>解析ソフトウェアで保存された記録を開き、1つのセルから刺激されたアクション電位を含む平均10スイープを開きます。</li>
	<li>Fオフセット を表すベースライン信号の平均を取り、平均されたトレースからこれを減算します。</li>
	<li>次の式∆F/F0を計算します(Fは蛍光を測定し、F0は拡張蛍光です)。 <img alt="Equation 3" src="/files/ftp_upload/61890/61890equ03v2.jpg">
</li>
	<li>トレースベースライン(拡張期)と対象領域(AP)を特定し、所望の心臓行動電位パラメータを測定する。これには、50%(APD50)および90%(APD90)の再分極の減衰時間が含まれますが、これらに限定されません。</li>
	<li>この単一のセルからスプレッドシート ソフトウェアにデータをエクスポートします。</li>
	<li>すべての録画について、手順 6.1 ~ 6.5 を繰り返します。適切な不対検定または分散分析で結果を評価します。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61890/61890fig2v2.jpg"> 図2:読み込みおよび画像取得プロトコル(A) iPSC-CM およびネイティブ心筋細胞用の完全な VF2.1Cl 負荷プロトコルのフローチャート。(B) ビームスプリッタ(BS)と、このプロトコルで使用されるフィルタ構成の簡略化された概略で、膜貫通電圧の変化に応じてVF2.1Cl発光の励起および検出を行う。 <a href="https://www.jove.com/files/ftp_upload/61890/61890fig2v2large.jpg" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a>

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電気生理学的モデリングや心臓薬スクリーニングに適した単離されたiPSC-CMから詳細なAPプロファイルを容易に取得するための基本的なプロトコルについて説明する。私たちは、インジケーター機能と方法論的忠実性の両方を示唆する、まばらなシードのiPSC-CMから定期的で堅牢なAPを検出します。
iPSCリプログラミングのための商業的方法論の広いスペクトルと心臓分化プ?...

心筋細胞の電気生理学的モデリングと心臓薬物スクリーニングのための効率的なプラットフォームの構築は、様々な不整脈障害の治療戦略の開発に不可欠です。誘導多能性幹細胞(iPSC)技術の急速な拡大は、単離患者由来心筋細胞(iPSC-CM)を用いたヒト疾患モデリングおよび薬理学的調査への有望な進出を生み出した。パッチクランプ(電流クランプ)を介してこれらの細胞の電気生理学的特徴付けのための「ゴールドスタンダード」技術は、作用電位(AP)形態および持続時間を定量化することができるが、この方法は非常に複雑で遅く、高スループットデータ取得1に適していない。iPSC-CMは、成人の天然心筋細胞2と比較して、拡張期膜電位が増加し、リーク電流が増加すると定期的に報告されている。iPSC-CMで観察される小さな細胞サイズおよび減少した膜容量は、電流クランプ技術を使用する際に系統的な誤差を生じさせる可能性があり、これらの偏差を説明する可能性が示唆される3。iPSC-CMプラットフォームの有用性を最大限に高めるためには、iPSC-CMで膜貫通電圧変化を単一のセルレベルで特性化する際のスループットとデータ精度を高めるため、追加の方法が重要です。
電圧感受性色素(VSD)は、従来の技術4と比較して、心臓AP動態のより速く、非侵襲的かつ同等の分析を提供する方法として提案されてきた。最近の研究では、レシオメトリック電圧感受性プローブフォトメトリーの適合性が、心臓AP5を正確に定量化することが実証されました。さらに、光学的な光測定のアプローチを容易にスケールアップする能力は、治療薬の開発に重要な大規模な心毒性スクリーン(例えば、CiPA)にこの技術を貸す。微小電極アレイと電圧感知光学技術を用いたブラインドマルチサイト研究における標準化された心毒性プロトコルの開発は、このアプローチの重要な価値を実証した6。
多くのポテンショメトリック色素は市販されており、新しいプローブの継続的な合成開発は、様々な心臓および神経構造にわたってその有効性を合理化するためのエキサイティングな可能性を示しています。理想的なVSDは、容量性負荷、光漂白および細胞毒性の低下を示しながら、運動および感受性を増強する。最近合成されたVF2.1Cl(FluoVolt)は、新しい電圧蛍光(VF)ファミリー7の他のメンバーが共有する、その新しいワイヤベースの分子構造に大きく起因するこれらの有益な特性の多くを表す。単純なプローブが分子的および電気的に化学接合する一般的なエレクトロクロミックVSDとは対照的に、この色素は、電子リッチドナーと修飾フルオロフォア(FITC)を組み合わせた受動的に挿入された膜スパン合成ワイヤーで構成されています。機械の詳細は 図 1に示されています。この色素は、膜電圧変動に対する優れた感度を示し、同等の速度で他の一般的なプローブに見られる〜10%とは対照的に、100mV当たりの発光強度の27%の変化を示す7。さらに、ワイヤベースのPeTシステムは、最小限の電気的干渉と細胞容量負荷のわずかな変化を生じるセルラー電界と直接相互作用しません。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61890/61890fig01.jpg"> 図1:VF2.1Cl色素の化学的、スペクトル的および機械的なパラメータ。(A)VF2.1Clの化学的構造. 注意すべき分子的特徴は、フェニレンビニレン分子線内の複数のアルキル基を含み、この細胞膜への挿入を容易にする。FITCプローブに結合した負の帯電スルホン酸基は、細胞外表面のフッ素酸安定化を保証し、脂質二重層の電界に対する垂直挿入に近い助けとなります。(B)標的細胞の細胞膜に埋め込む垂直VF2.1Clの簡略図である。(C)VF2.1Cl色素の吸収および発光スペクトル。スペクトルは、標準的なFITCおよびGFPプローブのそれと同じです。(D) VF2.1Clの作用の機械モードの描写。安静状態(過分極)では、負の細胞内電圧は、rostralフルオロフォアに向かって自由電子を駆動します。電子の存在量は光励起後の励起状態から外れる経路として光誘導電子移動(PeT)が好ましいこと、蛍光を効果的に消光することを保証する。これに対して、脱分極膜電位は、光学励起時に蛍光を好む下向きの電子移動に影響を与える。得られた蛍光応答は膜電圧に直線的に関連しており、細胞電生理学的運動に関する詳細な時間情報を収集するために正確に利用することができる。(E)VF2.1Clを搭載したレポリン心筋細胞の470nm(下)画像における代表的な明視野(上)および蛍光(F)単一の装填された心筋細胞のZスタック。矢印は、細胞膜へのVF2.1Clの明確な局在の領域を示す。画像は、50 μmのピンホールパターンを持つX-lightv3回転ディスク共焦点ヘッドからなる回転ディスク共焦点システムで取得されました。LDI-7イルミエーター;Prime95BカメラとPlanApoラムダ100xの目的。スケールバー:20 μm.<a href="https://www.jove.com/files/ftp_upload/61890/61890fig01large.jpg" target="_blank">この図の大きなバージョンを表示するには、ここをクリックしてください。</a>
VF2.1Clに結合したFITCプローブは、標準フィルタ構成およびGFPフィルタ構成下で効果的に使用できることを保証し、単一のチャネル取得システムのみが必要であり、どちらも蛍光イメージングプラットフォームの共通の特徴です。この色素を用いた高密度ヒトiPSC-CM単層の解析は、最近8、9、10、11と報告されている。我々のプロトコルは、緻密な一層の電気的およびパラクリンの影響によって妨げられない単一の孤立したiPSC-CMの調査と、複雑な共焦点または広視野イメージングの取り決めとは対照的に、手頃でカスタマイズ可能なフォトメトリーシステムの使用のために、これらの研究とは異なる。
ここでは、孤立したヒトiPSC由来の心筋細胞およびネイティブ心筋細胞からの堅牢な光学APの迅速な取得と分析のためのプロトコルについて説明 します(補足ファイルを参照)。我々は、単一細胞のフォトメトリー測定のための最先端のプラットフォームのカスタマイズ可能な状態と結合されたVF2.1Clを使用する。これらの実験プロトコルは、大学医療センターゲッティンゲン(No. 10/9/15)の倫理委員会によって承認されています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25 Trypsin EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement&#160;</td>
			<td>Gibco&#160;</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Carl Roth&#160;</td>
			<td>HN04.2</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose anhydrous&#160;BioChemica</td>
			<td>ITW Reagents</td>
			<td>A1422</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Gibco&#160;</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoVolt Membrane Potential Kit&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>F10488</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth&#160;</td>
			<td>HN77.4</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lamanin</td>
			<td>Sigma-Aldrich</td>
			<td>114956-81-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel&#160;</td>
			<td>BD</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>9265.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nifedipine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>21829-25-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen&#160;</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor Y27632</td>
			<td>Stemolecule</td>
			<td>04-0012-10</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium&#160;</td>
			<td>Gibco&#160;</td>
			<td>61870010</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>15040033</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>495LP Dichroic Beamsplitter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B Amplifier&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Circle Coverslips, Thickness 0</td>
			<td>Thermo Scientific</td>
			<td>CB00100RA020MNT0</td>
			<td></td>
		</tr>
		<tr>
			<td>Digidata 1550B</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dual OptoLED Power Supply&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ET470/40x Excitation Filter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ET535/50m</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Etched Neubauer Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Cubes&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IX73 Inverted Microscope&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MonoLED</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiport Adaptors&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
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		<tr>
			<td>Myopacer Cell Stimulator&#160;</td>
			<td>IonOptix</td>
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			<td>Optomask Shutter&#160;</td>
			<td>Cairn Research&#160;</td>
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			<td>Optoscan System Controller</td>
			<td>Cairn Research&#160;</td>
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			<td>PH-1 Temperature Controlled Platform&#160;&#160;</td>
			<td>Warner Instruments&#160;</td>
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			<td>Photomultiplier Detector&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
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		<tr>
			<td>PMT Amplifier Insert</td>
			<td>Cairn Research&#160;</td>
			<td></td>
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			<td>PMT Supply Insert</td>
			<td>Cairn Research&#160;</td>
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			<td>RC-26G Open Bath Chamber&#160;</td>
			<td>Warner Instruments&#160;</td>
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			<td>SA-OLY/2AL Stage Adaptor&#160;</td>
			<td>Olympus&#160;</td>
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			<td>T565lpxr Dichroic Beamsplitter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
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			<td>T660lpxr Dichroic Beamsplitter</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
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		<tr>
			<td>TC-20 Dual Channel Temperature Controller&#160;</td>
			<td>npi Electronic</td>
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			<td>UPLFLN 40X Objective</td>
			<td>Olympus&#160;</td>
			<td></td>
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		<tr>
			<td>USB 3.0 Colour Camera&#160;</td>
			<td>Imaging Source</td>
			<td></td>
			<td></td>
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			<td>Software</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Clampex 11.1</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampfit 11.1</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IC Capture 2.4&#160;</td>
			<td>Imaging Source&#160;</td>
			<td></td>
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		<tr>
			<td>Prism 8</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes

従来の細胞内微小電極技術は、心筋細胞電気生理学を定量する、極めて複雑で、労働集約的であり、典型的には低スループットで行われる。誘導多能性幹細胞(iPSC)技術の急速かつ継続的な拡大は、心血管研究における新しい標準を提示し、単一細胞レベルでの電気生理学的データのスループットを向上させるために代替方法が必要になりました。VF2.1Clは、膜電位の変動に対する急速な単一チャネル、高い強度応答を提供する最近得られた電圧感受性色素です。他の既存の電圧指標よりも優れた運動学を有し、従来の微小電極技術と同等の機能データを利用できます。ここでは、モジュール式で手頃な価格のフォトメトリーシステムを用いて、外部ペースのヒトiPSC由来心筋細胞における、単純化された非侵襲的作用電機の特性を実証する。

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61890/61890fig03.jpg"> 図3:単離した天然型心筋細胞およびヒト誘導多能性幹細胞由来心筋細胞(iPSC-CM)の光学作用電位(AP)プロファイル。(A)APD50およびAPD90の平均±SEMを有する単一のマウス心筋細胞(中央)の代表的な光学AP(n = 7、右)。(B) APD50およびAPD90の平均±SEM?...

Watch this Scientific Journal Video about Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

ここでは、高速モジュラーフォトメトリーシステムを用いた誘導多能性幹細胞由来心筋細胞からの作用電位の光学的獲得および特性評価について述べている。

ヒト人工多能性幹細胞由来心筋細胞における単細胞光学作用電位測定

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

single-cell-optical-action-potential-measurement-human-induced

Research

JoVE Journal

Bioengineering

4.0K ビュー.  University Medical Center Goettingen, Goettingen, Germany. ここでは、高速モジュラーフォトメトリーシステムを用いた誘導多能性幹細胞由来心筋細胞からの作用電位の光学的獲得および特性評価について述べている。

ビデオ: Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

マウスの心臓の活動電位およびカルシウムトランジェントの光学的マッピング

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

生物工学

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

開発、展開、および<em生体内で&gt;</em&gt;ヒト胚性幹細胞（ヒトES細胞）からヒトNK細胞のモニタリングとや人工多能性幹細胞（性IPSC）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

筋肉再生のマウスモデルにおける誘導多能性幹細胞由来メソアンジオ芽細胞様筋原性前駆細胞の移植

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

再発作用ポテンシャル記録のためのマルチウェルマイクロ電極アレイ上のヒトiPSC由来心筋細胞ネットワーク

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

化学的に定義された条件下でのヒト人工多能性幹細胞からの成熟した腎臓ポドサイトの誘導分化

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

ヒト人工多能性幹細胞から改変された同質遺伝子腎臓糸球体チップ

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

2次元ヒト幹細胞由来心筋細胞における心収縮性調節療法の評価

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

多能性幹細胞からのヒト血管オルガノイドの作製

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

ヒト誘起多能性幹細胞由来心臓スフェロイドの作製、ハイスループットスクリーニング、バイオバンキング

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

成熟ヒト人工多能性幹細胞由来心筋細胞単分子膜を用いたハイスループット心毒性スクリーニング

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...