この研究は、デューク大学のプラット工学部、デューク医科大学腎臓学科、デューク大学医学部の椅子研究賞、バロウズウェルカム基金PDEPキャリア移行 アドホック 賞によって支援されました.M。M.Bは、国立科学財団大学院研究フェローシッププログラムの支援を受けています。DU11幹細胞ラインを寛大に提供してくれたBursac Labと、デューク大学のヴァルゲーゼラボが組織培養施設を一時的に共有してくれたことに感謝します。この出版物は、60歳の誕生日 を祝って、マサチューセッツ工科大学のノバルティス化学教授ローラ・L・キースリング教授に捧げられています。

1. 試薬の調製
<ol><li>HiPS細胞培養基底培地で解凍した5x hiPS細胞培養培地(CCM)サプリメントを希釈し、hiPS CCMの1x溶液を得た。 注:冷凍5x hiPS CCMサプリメントは、理想的には一晩4°Cで、ゆっくりと解凍プロセスを必要とします。1x hiPS CCMのアリコートは-20°Cで6ヶ月まで保存することができます。</li>
	<li>hiPS細胞培養用基膜(BM)マトリックス1コーティングプレートの調製:4°Cで氷上でBMマトリックス1を一晩解凍する。解凍したら、メーカーが提案した適切な希釈因子を持つアリコートを準備します。 注:通常、アリコートは、50 mL円錐管内の冷たいDMEM/F12の25 mLで、BMマトリックス1を完全に溶解し、残留結晶の形成を避けるために完全に混合して、その後の希釈のために準備されます。</li>
	<li>BMマトリックス1溶液の1mLを6ウェルプレートのウェルに移し、37°Cで1〜2時間、または4°Cで最低24時間インキュベートし、パラフィンフィルムで包みます。BMマトリックス1-被覆されたプレートは、4°Cで最大2週間保存することができます。</li>
	<li>基質膜(BM)マトリックス2の調製-コーティングされた版:9 mLの無菌蒸留水のBMマトリックス2の適切な量を希釈して5μg/mLの最終濃度を作製する。BMマトリックス2溶液700μLを12ウェルプレートのウェルに加え、室温でプレートを2時間または4°Cで一晩インキュベートします。</li>
	<li>BMP7、アクティビンA、VEGFの各100 μg/mLストック溶液を次のように準備します:0.1%(wt/vol)BSAを含む無菌蒸留水でBMP7を再構成し、アクチビンAとVEGFを0.1%(wt/vol)BSAを含む無菌PBSで別々に再構成します。頻繁な凍結融解サイクルを避けるために、各ストック溶液から100 μLのアリコートを準備し、-20°Cで最大6ヶ月間保管します。</li>
	<li>3.079 mLの無菌蒸留水に10mgのY27632を溶解して、Y27632の10mMストック溶液を調製します。アリコート100 μLは、在庫から-20°Cで6ヶ月間保管します。</li>
	<li>滅菌DMSOの143.4 μLにCHIR99021の2mgを溶解し、30mMストック溶液を調製します。5 μLのアリコートを準備し、-20°Cで最大1ヶ月間保管してください(またはメーカーの推奨に従ってください)。</li>
	<li>3.33 mL滅菌DMSOでオールトランス レチノイン酸の10mgを溶解する。500 μL アリコートを準備し、-20 °Cで6ヶ月間保管します。</li>
</ol>2. 文化メディアの作成
<ol><li>グルタミンサプリメントを含むDMEM/12の適切な容積で、対応するストック溶液を最終濃度100 ng/mL ActR99021、10 μM Y27632、および1x B27無血清サプリメントに再構成して中皮分化培地を調製します。 注:中皮分化培地は、分化ステップの前に、実験の規模に適したボリュームで新たに調製する必要があります(通常、50 mLの培地は2つの12ウェルプレートに適しています)。</li>
	<li>グルタミンサプリメントを用いて、DMEM/F12の最終濃度100 ng/mL BMP7、3 μM CHIR99021、および1x B27のB27血清フリーサプリメントに対応するストック溶液を再構成することにより、中間中皮分化培地を調製します。 注:必要に応じて、培地は1%(vol/vol)ペニシリンストレプトマイシンで補うことができます。グルタミンサプリメントで DMEM/F12 を使用して、媒体の量を調整します。.この媒体は大きいバッチで準備することができる。しかし、凍結融解サイクルを繰り返さないように、小さなアリコート(例えば、50 mL円錐管で45 mL)に保存することをお勧めします。この培地は-20°Cで3ヶ月間保存でき、使用前に一晩4°Cで解凍することができます。</li>
	<li>最終濃度100 ng/mL BMP7、100 ng/mLアクチビンA、50 ng/mL VEGF、3 μM CHIR99021、1x B27血清無血清サプリメント、および0.1 μM全トランスレチノイン酸をDMEM/F12型食品で補間して再コンセプトして、ポドサイト誘導培地を調製します。媒体を光から守る(例えば、ホイル紙で容器を包むことで)。 注:この培地は、大きなバッチで調製し、3ヶ月まで-20°Cで暗闇の中に保存することができます。冷凍アリコートは、使用前に4°Cで一晩解凍する必要があります。</li>
	<li>DMEM/F12に10%(vol/vol)熱不活化FBSを加え、滅菌条件下でフィルターを加えて25 mLのトリプシン中和溶液を調製します。</li>
	<li>幹細胞由来ポドサイトの分化後のメンテナンスのために、製造者のガイドラインに従って基礎培地にサプリメントを添加してポドサイト維持培地でコンプリート培地を準備し、4°Cで最大2週間保存します。</li>
</ol>3. ハイプス細胞培養培地を用いたフィーダーフリーHiPS細胞培養
<ol><li>BMマトリックス1の残留溶液をプレコートされたプレートから吸引し、1〜2mLの加温DMEM/F12でウェルを3回洗浄した。</li>
	<li>吸引はhiPS細胞からhiPS CCMを使用し、温められたDMEM/F12で細胞を3回すすいでください。1 mLの温細胞剥離液を加え、37°Cで1分間インキュベートして細胞の解離を助けます。組織培養顕微鏡で細胞を目視検査し、細胞コロニーの縁部が丸みを帯びているように見えるようにし、細胞外着液を細胞から素早く吸引する(細胞コロニーがプレートに付着していることを確認する( ゆるやかに見えるが、細胞コロニーがプレートに付着していることを確認する)。DMEM/F12で細胞を1回緩やかにすすいで、細胞剥離液を除去します。</li>
	<li>3 mLのhiPS CCMを、細胞剥離液で処理したhiPS細胞(6ウェルプレートの各ウェル)に加えます。セルリフターを使用してコロニーを擦り、ピペット細胞懸濁液を上下に静かにして緩やかに接着した細胞を取り除きます。プレートを十分に洗い、すべての細胞が収穫されるようにします。 注:細胞に余分なせん断を避けるために5 mLピペットの使用をお勧めします。</li>
	<li>1ウェルあたり2 mLのhiPS CCMを含む新しいBMマトリックス1コーティング6ウェルプレートの各ウェルに、細胞懸濁液の0.5 mLを移します。ウェル内の細胞コロニーを分配し、5%CO2インキュベーターで37 °Cでインキュベートするために、図8の方法でプレートを移動します。細胞が分化実験に通過または使用される準備ができるまで、毎日培地をリフレッシュします(約70%の合流率)。 注:iPSCのルーチンパスエージングのための理想的なコロニーサイズはhiPS CCM(ROCK阻害剤なし)を使用してフィーダーフリー条件下で200-500 μmの間です。解離酵素またはバッファーを使用した処理中に細胞を個別化した場合、hiPS CCMをROCK阻害剤(例えば、10 μM Y27632)で補い、細胞の生存率を向上させることができます。</li>
</ol>4. 中皮細胞へのhiPS細胞の分化(0-2日目)
<ol><li>HiPS細胞培養は指数成長段階にあるが(通過後約4日以内、約70%の合流率)、コロニーの縁の内側および周囲に自発的に分化した細胞の存在を視覚的に検査する。必要に応じて、分化の無菌的に擦り落ち領域。</li>
	<li>hiPS細胞からHIPS CCMを吸引し、温かいDMEM/F12で細胞を3回すすます。37°Cで10分間酵素フリー細胞解離バッファーを1 mLで培養し、顕微鏡下で解離を確認します。異なるhiPS細胞株間の固有の違いのために、細胞解離バッファーの実際のインキュベーション時間は、所定の細胞株について決定されなければならない。</li>
	<li>セルリフターでウェルを静かにこすり取り、ゆるやかに接着した細胞を取り除き、細胞懸濁液を15 mL円錐形チューブに移し、続いて数回上下にピペットを作ってHiPS細胞を個体化します。<ol>
<li>セル懸濁液を、温かいDMEM/F12で15mLの体積に、遠心分離機を室温で290 x g で5分間持ちます。</li>
		<li>優しく上清を吸引し、残存BMマトリックス1および解離緩衝成分を除去するために遠心分離の別のラウンドのために暖かいDMEM / F12で細胞を再懸濁します。</li>
	</ol>
</li>
	<li>上清を吸引し、上述のように中皮誘導培地の1mLで細胞を再懸濁する。1 x 105 細胞/mLの濃度を達成するために必要な中皮分化培地の適切な体積を決定するために、ヘモサイトメーターまたはコールターカウンターを使用して細胞の総数を数えます。</li>
	<li>BMマトリックス2コーティングプレートからのECM溶液を吸引し、温かいDMEM/F12でプレートを2回リンスします。数回ピペットを使用して、hiPS細胞の懸濁液を軽く混ぜます。セル懸濁液1mLをBMマトリックス2コーティング12ウェルプレートのウェルそれぞれに移し、プレートを穏やかに振って細胞をより均等に分配します。</li>
	<li>プレートを37°Cで5%CO2インキュベーターでインキュベートします。翌日中皮誘導培地をリフレッシュします。 注:2日後、hiPS細胞由来の中皮細胞は中間中皮誘導の準備が整いました。</li>
</ol>5. 中間中半分へのhiPS細胞由来中皮細胞の分化(2-16日目)
<ol><li>分化プロトコルの2日目には、中皮誘導媒体を吸引し、1 mL当たり1mLの中間中期中期誘導媒体で補充する。</li>
	<li>代謝活性細胞の成長因子と小分子の正確な閾値を維持するために、毎日培地をリフレッシュします。実質的な細胞増殖およびメディア栄養素の急速な枯渇(メディアの黄変によって示される)がある場合、中間中皮分化培地の体積は、12ウェルプレートのウェル当たり1.3mLに増加させることができる。</li>
	<li>培養細胞は、中間中皮細胞を得るためにさらに14日間を行った。16日目までに、これらの細胞は後で使用するために凍結保存することができる。</li>
</ol>6. hiPS細胞由来中間中中軟体細胞のポドサイトへの分化(16日~21日目)
<ol><li>中間中皮細胞を温かいDMEM/F12でリンスし、37°Cで3分間0.05%トリプシン-EDTAのウェル当たり0.5 mLで細胞をインキュベートします。細胞の解教が始まっていることを確認するために目視検査を行います。</li>
	<li>細胞リフターを使用して細胞をスクレープし、1,000 μLピペットチップを使用して細胞懸濁液を数回使用して、個別化された(または小さな塊の)細胞を得る。 注: この段階では、集約されたセルがプロトコルのタイムライン内で末端に分化された表現型を取得できない可能性があるため、セルが完全に解約されていることを確認してください。</li>
	<li>トリプシン中和溶液のウェルあたり約2 mLを加えてトリプシンの活性を停止する。</li>
	<li>細胞を50 mL円錐形チューブに移し、DMEM/F12を使用して体積を最大50 mLにし、室温で201 x g で5分間細胞懸濁液を遠心分離します。</li>
	<li>上清を吸引し、ポドサイト誘導培地中の細胞を再懸濁する。最適な結果を得るには、12ウェルプレートの約100,000個の細胞/ウェルの最終播種密度を確保してください。セル懸濁液をBMマトリックス2コーティングプレートに加え、プレートを穏やかに振って細胞をより均等に分配します。</li>
	<li>細胞を37°Cおよび5%CO2でインキュベートし、培地を毎日5日間までリフレッシュし、21日目までにポドサイトを得た。 注:得られたhiPS細胞由来ポドサイトは、ポドサイト維持培地を含む完全な培地を使用することにより、培養で2〜4週間維持することができます。一度ポドサイト維持培地に入ると、細胞は1日おきに供給することができ、その後の研究や下流の分析に使用することができます。</li>
</ol>

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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+and+robust+induction+of+nephrogenic+intermediate+mesoderm+from+human+pluripotent+stem+cells.">Monitoring and robust induction of nephrogenic intermediate mesoderm from human pluripotent stem cells.</a> Nature Communications. 4, 1367 (2013).</li><li>Reya, T., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signalling+in+stem+cells+and+cancer.">Wnt signalling in stem cells and cancer.</a> Nature. 434, 843-850 (2005).</li><li>Clevers, H., Nusse, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt&#946;-Catenin+Signaling+and+Disease.">Wnt&#946;-Catenin Signaling and Disease.</a> Cell. 149, 1192-1205 (2012).</li><li>Ciampi, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+podocytes+from+human+induced+pluripotent+stem+cells.">Generation of functional podocytes from human induced pluripotent stem cells.</a> Stem Cell Research. 17, 130-139 (2016).</li><li>Fuente Mora, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+Podocyte+and+Proximal+Tubule-Like+Cells+from+a+Mouse+Kidney-Derived+Stem+Cell+Line.">Differentiation of Podocyte and Proximal Tubule-Like Cells from a Mouse Kidney-Derived Stem Cell Line.</a> Stem Cells and Development. 21, 296-307 (2011).</li><li>Chuah, J. K. C., Zink, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-derived+kidney+cells+and+organoids:+Recent+breakthroughs+and+emerging+applications.">Stem cell-derived kidney cells and organoids: Recent breakthroughs and emerging applications.</a> Biotechnology Advances. 35, 150-167 (2017).</li><li>Becker, G. J., Hewitson, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+chronic+kidney+disease:+useful+but+not+perfect.">Animal models of chronic kidney disease: useful but not perfect.</a> Nephrology Dialysis Transplantation. 28, 2432-2438 (2013).</li></ol>

S.Mは、多能性幹細胞からの腎臓ポドサイトの生成方法の特許出願中の著者です(米国特許出願14/950859)。残りの著者は、彼らが競合する利益を持っていないと宣言します。

本報告では、hiPS細胞からの腎臓糸球体ポドサイトの生成プロトコルについて述べている。hiPS細胞由来ポドサイトは、成熟した腎臓ポドサイト表現型13に関連する形態学的および分子的特徴を示す。これまでの刊行物では、hiPS細胞由来ポドサイトが、透過性マイクロ流体臓器オンチップ装置8,11で糸球体微小血管内皮細胞と共培養?...

ヒト多能性幹細胞培養の進歩は、再生医療、疾患モデリング、および薬物スクリーニングに革命をもたらす準備ができているので、研究者は、人体内のほぼすべての細胞型を得るように設計することができる再生可能でスケーラブルな生物学的材料の供給源を提供する1。この方法は、他の方法では入手が困難な特殊な機能型のセル型を派生する場合に特に役立ちます。ヒト人工多能性幹細胞(hiPS)細胞2、3、4、5は、体細胞起源と個別化医療の可能性から特に魅力的です。しかし、hiPS細胞から他の細胞系統を導き出す方法の開発は、不十分に定義された培養条件を頻繁に使用し、不十分な培養条件を使用し、6,7の異種細胞集団の低効率および非特異的生成を招くため、依然として困難である。
ここで提示される、化学的に定義された条件下で特異性および高効率のhiPS細胞からの成熟した腎臓ポドサイトの導出のための方法である。細胞微小環境内での複数因子の役割を考慮することにより、細胞培養培地に示される可溶性因子の最適化や、細胞外マトリックス成分や接着基材などの不溶性因子の最適化を含む幹細胞分化戦略が開発されました。ポドサイトの発達と機能におけるインテグリンシグナル伝達の重要性を考えると、細胞表面上のインテグリン受容体の発現を最初に検討した。β1インテグリンはhiPS細胞のみならず、中皮細胞及び中間中皮細胞8、9、10を含む誘導体においても高発現した。その後の実験では、β1インテグリン(ラミニン511またはラミニン511-E8フラグメントを含む)に結合するリガンドが、以下に記載の可溶性誘導体と組み合わせて使用した場合に、hiPS細胞のポドサイトへの接着および分化を支持することが確認された。
細胞系統の誘導は、アクチビンA、CHIR99021、およびY27632ロック阻害剤を含む培地の存在下で2日間ラミニンコーティングされた表面上で培養されたhiPS細胞が、初期のメソダームマーカーHAND1、グースコイド、および小頭体8,11を発現する細胞に分化できることを最初に確認することによって開始された。骨形態形成タンパク質7(BMP-7)およびCHIR99021を添加した培地を用いた14日間の中皮細胞の治療は、ニーフロン前駆細胞マーカーウィルム腫瘍1(WT1)を発現する中間中足細胞の誘導を可能にし、奇数スキップ関連タンパク質1(OSR1)8、11、およびペア化したタンパク質22(PAX)を用いた。成熟した腎臓糸球体の細胞を導出するために、中間中期細胞をBMP-7、アクチビンA、血管内皮増殖因子(VEGF)、全トランスレチノイン酸、およびCHIR99021からなる新規培地で4〜5日間処理した。フローサイトメトリーおよび免疫染色は、得られた細胞の90%> が成熟した腎臓ポドサイト8、11、13の分子、形態学的、および機能的特徴を示すことを確認するために使用した。これらの特性には、一次および二次的な足のプロセスの開発が含まれます。SYNPO、PODXL、MAF、EFNB28を含むポドサイト系統特異的遺伝子の発現と、ポドシン、ネフリン、およびWT114、15、16を含むタンパク質の発現。さらに、hiPS細胞由来ポドサイトは、下流実験のタイミングにさらなる柔軟性を提供する市販の培地8、11を用いることによって、最大4週間の培養液中で維持できることが分かった。hiPS-podocytesの純度を決定するために使用されるフローサイトメトリーパネルの詳細については、当社の前の出版物11を参照してください。

<table>
	<tbody>
		<tr>
			<td>Cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DU11 human iPS cells</td>
			<td></td>
			<td></td>
			<td>The DU11(Duke University clone #11) iPS cell line was generated at the Duke University iPSC Core Facility and provided to us by the Bursac Lab at Duke University. This line has been tested for mycoplasma and was last karyotyped in July 2019 by our lab, and found to be karyotypically normal.</td>
		</tr>
		<tr>
			<td>Growth Factors and Media Supplements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>All-trans retinoic acid (500 mg)</td>
			<td>72262</td>
			<td>Stem Cell Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 serum-free supplement</td>
			<td>17504044</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>04-0004</td>
			<td>Stemgent</td>
			<td>May show lot-to-lot variation</td>
		</tr>
		<tr>
			<td>Complete Medium Kit with CultureBoost-R</td>
			<td>4Z0-500-R</td>
			<td>Cell Systems</td>
			<td>Podocyte maintenance media</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>12634028</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 with GlutaMAX supplement</td>
			<td>10565042</td>
			<td>Thermo/Life Technologies</td>
			<td>DMEM/F12 with glutamine supplement</td>
		</tr>
		<tr>
			<td>Heat-inactivated FBS</td>
			<td>10082147</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human activin A</td>
			<td>PHC9564</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BMP7</td>
			<td>PHC9544</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Human VEGF</td>
			<td>PHC9394</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 medium</td>
			<td>05850</td>
			<td>Stem Cell Technologies</td>
			<td>hiPS cell culture media (CCM)</td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin, liquid (100&#215;)</td>
			<td>15140-163</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Y27632 ROCK inhibitor</td>
			<td>1254</td>
			<td>Tocris</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488&#8211; and Alexa Fluor 594&#8211;conjugated secondary antibodies</td>
			<td>A32744; A32754; A-11076; A32790</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Brachyury(T&#65289;</td>
			<td>ab20680</td>
			<td>Abcam</td>
			<td></td>
		</tr>
		<tr>
			<td>Nephrin</td>
			<td>GP-N2</td>
			<td>Progen</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT4</td>
			<td>AF1759</td>
			<td>R&#38;D Systems</td>
			<td></td>
		</tr>
		<tr>
			<td>PAX2</td>
			<td>71-6000</td>
			<td>Invitrogen</td>
			<td></td>
		</tr>
		<tr>
			<td>WT1</td>
			<td>MAB4234</td>
			<td>Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM Molecules</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iMatrix-511 Laminin-E8 (LM-E8) fragment</td>
			<td>N-892012</td>
			<td>Iwai North America</td>
			<td>Basement membrane (BM) matrix 2</td>
		</tr>
		<tr>
			<td>Matrigel hESC-qualified matrix, 5-mL vial</td>
			<td>354277</td>
			<td>BD Biosciences</td>
			<td>Basement membrane (BM) matrix 1. May show lot-to-lot variation</td>
		</tr>
		<tr>
			<td>Enzymes and Other Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>A1110501</td>
			<td>Thermo/Life Technologies</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>A9418</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>D2438</td>
			<td>Sigma-Aldrich</td>
			<td>DMSO is toxic. Should be handled in chemical safety hood</td>
		</tr>
		<tr>
			<td>Enzyme-free cell dissociation buffer, Hank&#8217;s balanced salt</td>
			<td>13150016</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol solution, 70% (vol/vol), biotechnology grade</td>
			<td>97065-058</td>
			<td>VWR</td>
			<td>Ethanol is flammable and toxic</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>431097</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>28906</td>
			<td>Thermo/Life Technologies</td>
			<td>PFA should be handled in a chemical fume hood with proper personal protection equipment, including gloves, lab coat, and safety eye glasses. Avoid inhalation and contact with skin.</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>14190-250</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Distilled Water</td>
			<td>15230162</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>97062-208</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.05%</td>
			<td>25300-120</td>
			<td>Thermo/Life Technologies</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirating pipettes, individually wrapped</td>
			<td>29442-462</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-15R Centrifuge</td>
			<td>B99516</td>
			<td>Beckman Coulter</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tube, 15 mL</td>
			<td>352097</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical centrifuge tube, 50 mL</td>
			<td>352098</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryoboxes</td>
			<td>3395465</td>
			<td>Thermo/Life Technologies</td>
			<td>For storing frozen aliquots</td>
		</tr>
		<tr>
			<td>EVOS M7000</td>
			<td>AMF7000</td>
			<td>Thermo/Life Technologies</td>
			<td>Flourescent microscope used to acquire images of fixed and stained iPS cells and their derivatives</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>100503-092</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell VIOS 160i CO2 incubator</td>
			<td>51030403</td>
			<td>Thermo/Life Technologies</td>
			<td>For the routine culture and maintenace of iPS cells and their derivatives</td>
		</tr>
		<tr>
			<td>Inverted Zeiss Axio Observer equipped with AxioCam 503 camera</td>
			<td>491916-0001-000(microscope) ; 426558-0000-000(camera)</td>
			<td>Carl Zeiss Microscopy</td>
			<td>Used to acquire phase contrast images of live iPS cells and their derivatives at each stage of podocyte differentiation</td>
		</tr>
		<tr>
			<td>Kimberly-Clark nitrile gloves</td>
			<td>40101-346</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, large</td>
			<td>21905-049</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes, small</td>
			<td>21905-026</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>P10 precision barrier pipette tips</td>
			<td>P1096-FR</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P100 barrier pipette tips</td>
			<td>P1125</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 barrier pipette tips</td>
			<td>P1126</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 barrier pipette tips</td>
			<td>P1121</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 barrier pipette tips</td>
			<td>P1122</td>
			<td>Denville Scientific</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 10 mL, individually wrapped</td>
			<td>356551</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 25 mL, individually wrapped</td>
			<td>356525</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette, 5 mL, individually wrapped</td>
			<td>356543</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Steriflip, 0.22 &#956;m, PES</td>
			<td>SCGP00525</td>
			<td>EMD Millipore</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Microcentrifuge Tubes</td>
			<td>1138W14</td>
			<td>Thomas Scientific</td>
			<td>For aliquoting growth factors</td>
		</tr>
		<tr>
			<td>Tissue culture&#8211;treated 12-well plates</td>
			<td>353043</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture&#8211;treated six-well plates</td>
			<td>353046</td>
			<td>Corning</td>
			<td></td>
		</tr>
		<tr>
			<td>VWR white techuni lab coat</td>
			<td>10141-342</td>
			<td>VWR</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-beveled cell lifter</td>
			<td>3008</td>
			<td>Corning</td>
			<td></td>
		</tr>
	</tbody>
</table>

guided differentiation of mature kidney podocytes from human induced pluripotent stem cells under chemically defined conditions

腎臓病は世界人口の10%以上に影響を及ぼし、連邦支出に数十億ドルの費用がかかります。腎臓病の最も重篤な形態および最終的な末期腎不全は、しばしば、内皮細胞および糸球体地下膜と一緒に機能する高度に特殊化された上皮細胞である糸球体ポドサイトの損傷によって引き起こされ、腎臓の濾過障壁を形成する。腎医学の進歩は、一次組織の利用が限られており、ポドサイトなどの機能性ヒト腎臓細胞の誘導のための堅牢な方法の欠如によって妨げられてきた。幹細胞などの再生可能な供給源からポドサイトを導き出す能力は、ヒト腎臓の発達と病気のメカニズムに関する現在の理解を進めるだけでなく、治療的発見のための新しいツールを提供するのに役立つ可能性があります。このプロトコルの目的は、高効率かつ特異性のヒト誘導多能性幹細胞(hiPS)細胞から、化学的に定義された条件下で成熟した、後の有糸球体を導き出す方法を開発することであった。この方法で生成されたhiPS細胞由来ポドサイトは、系統特異的マーカー(ネフリン、ポドシン、ウィルム腫瘍1を含む)を発現し、成熟した機能的ポドサイトに関連する特殊な形態学的特徴(一次および二次足プロセスを含む)を示す。興味深いことに、これらの特殊な特徴は、この分野で広く使用されている不死化ポドサイト細胞株には特に存在せず、本明細書に記載されているプロトコルは、ヒト腎臓生物学を研究するために典型的に使用される既存のポドサイト細胞株よりも発達的により成熟した表現型を有するヒト腎臓ポドサイトを産生することを示唆している。

このプロトコルの目的は、成熟したヒトポドサイトが化学的に定義された条件下でhiPS細胞から誘導できることを実証することであった。この原稿に記載されたデータは、DU11 hiPS細胞株17を用いて生成され、最初に試験され、マイコプラズマが含まれなかったことが判明した。染色体分析も行われ、細胞はカルヨタイプ正常であることが判明した。未分化DU11 hiPS細胞から、こ?...

Watch this Scientific Journal Video about Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions at JoVE.com

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

ここで提示されるのは、高効率(>90%)を有する誘導多能性幹細胞からのヒト腎臓ポドサイトの誘導のための化学的に定義されたプロトコルであり、遺伝子操作または亜集団選択とは無関係である。このプロトコルは、26日以内に望ましい細胞タイプを生成し、腎毒性試験および疾患モデリングに有用であり得る。

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 ...

guided-differentiation-mature-kidney-podocytes-from-human-induced

Research

JoVE Journal

Bioengineering

4.4K ビュー.  Duke University. ここで提示されるのは、高効率(>90%)を有する誘導多能性幹細胞からのヒト腎臓ポドサイトの誘導のための化学的に定義されたプロトコルであり、遺伝子操作または亜集団選択とは無関係である。このプロトコルは、26日以内に望ましい細胞タイプを生成し、腎毒性試験および疾患モデリングに有用であり得る。

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

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 ...

生物工学

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 ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

人間歯槽骨膜から骨膜由来細胞の骨形成促進活性に及ぼすビタミン D の間葉系幹細胞の分離

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

Xeno 無料条件下におけるさまざまな剛性とポリビニル アルコール Co イタコン酸ゲルのひと多能性幹細胞文化

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

腹部皮膚からの成人ヒト皮膚線維芽細胞の分離と非積分法を用いた人工多能性幹細胞の生成

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

ヒト多能性幹細胞からの成熟小脳オルガノイドのスケーラブルな生成と免疫染色による特徴付け

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

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

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

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 ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

スクリーニング用途のための線維芽細胞由来ヒト工学的結合組織

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

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 ...

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 ...