We thank Dr. Peter Zandstra, in whose laboratory this protocol was developed, and Drs. Mark Gagliardi and Gordon Keller who provided assistance in establishing the initial methods on which this process was based. Protocol development was supported by an Ontario Graduate Scholarship in Science and Technology to C.B. and a grant from the Heart and Stroke Foundation of Ontario to Peter Zandstra.

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J., Wrzesinski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+drug+toxicity+using+3D+spheroids+constructed+from+an+immortal+human+hepatocyte+cell.">Determination of drug toxicity using 3D spheroids constructed from an immortal human hepatocyte cell.</a> Toxicol. Sci. Off. J. Soc. Toxicol. 127, 403-411 (2012).</li><li>Wallace, L., Reichelt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+3D+culture+to+investigate+the+role+of+mechanical+signaling+in+keratinocyte+stem+cells.">Using 3D culture to investigate the role of mechanical signaling in keratinocyte stem cells.</a> Methods Mol. Biol. Clifton NJ. 989, 153-164 (2013).</li><li>Ungrin, M., O'Connor, M., Eaves, C., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+analysis+of+human+embryonic+stem+cells.">Phenotypic analysis of human embryonic stem cells.</a> Curr. Protoc. Stem Cell Biol. , (2007).</li><li>Bhattacharya, S., 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=High+efficiency+differentiation+of+human+pluripotent+stem+cells+to+cardiomyocytes+and+characterization+by+flow+cytometry.">High efficiency differentiation of human pluripotent stem cells to cardiomyocytes and characterization by flow cytometry.</a> J. Vis. Exp. JoVE. , e52010 (2014).</li><li>Nostro, M. 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Bioeng. 102, 493-507 (2009).</li></ol>

M.U. has a financial interest in the underlying microwell technology.

多能性幹細胞の効率的な心臓分化は、高度に可変なプロセスであることが観察されています。それは、異なる細胞株は、特定の細胞型への分化能のために変化する傾向を示すことは驚くべきことではないが、心臓の分化効率がレプリケート間劇的に変動することが観察された同じ細胞株6を使用して実行されます。ここで説明するプロトコルは、直接の集約あたりの入力セルの数を制御することによって、この変動の一つの大きな源に対処しています。さらに実行の間のばらつきを低減するためには、単一細胞継代のために適合HPSC線が多能性マーカーの発現頻度（ 例えば 、あるOct4、Nanogのに対して、より一貫性の多能性集団におけるHPSCの拡大とメンテナンス結果のこの形として、使用することをお勧めしますTRA-1-60、 等 。）。 ここに書かれたようなプロトコルがoのために1,000細胞の凝集体サイズを指定しますHES-2胚性幹細胞株からptimal心臓誘導。異なる細胞株にこのプロトコルを適用するためには、初期の凝集体サイズ画面は細胞株特異的な最適な凝集体サイズを決定するために実行することが重要です。それがここで直接従うべき手順には影響しませんが、我々は、凝集体サイズと全体的な細胞密度の変化は酸素供給に影響を与えることが期待されている読者を思い出させます。これは、下流のアプリケーションでは、関連する考慮なることがあります。さらに、アポトーシス細胞死は、単一細胞にhPSCsの解離時の関心事です。したがって、ROCK阻害剤は、マイクロウェルで強制細胞凝集の間に存在していることを確実にするために重要です。最後に、分化の4日目に凝集体は十分に誘導2培地で再懸濁する前に、誘導1中に存在し、トレースアクチビンAを除去するために洗浄することが重要です。分化の4日後に、アクチビンAはメソを犠牲にして内胚葉分化を促進しますダーム誘導20。 この技術の主な用途は、効率的な心臓分化を促進集計サイズをスクリーニングすることです。しかし、現在の技術の限界の一つは、マイクロウェルプレートを使用して臨床的に関連するレベルまで心筋の生産をスケールするために挑戦していることです。心臓分化のスケールアップは、典型的には、撹拌懸濁バイオリアクター21内のバルク培養条件で行われます。マイクロシステムは、効率的な心臓の誘導のための凝集体の大きさの許容範囲を決定するために使用された後、そのため、スケールアップする次のステップは、所望の細胞の凝集体のサイズを生成することができるバイオリアクターインペラー速度を決定することです。 集合ベース心臓分化のための他の方法に対するこの技術の重要な違いの1つは、それが同様に凝集体における内因性シグナルの効果を調節するに直接調査を可能にすることです集計13でhPSCsと誘導性/抑制性の組織型の共培養。調査のこれらのタイプは、大規模な心臓の製造のプロセス開発を知らせることができます。

インビトロ細胞培養でモダリティの数で行うことができるが、典型的には二次元の接着条件またはより完全にインビボ系において要約三次元懸濁状態のいずれかで行われます。その結果、三次元組織構築物を生成するための強力な方法を開発するための研究の多くの分野における成長傾向があります。細胞型およびプロセスは支持細胞外マトリックス（ECM）表面との密着性の信号を必要とするシナリオでは、三次元培養は、細胞が上又はマトリックス1を支持する外因性で培養される足場構築物を介して有効にすることができます。支持マトリックスへの付着を必要としない細胞と処理は、主にまたは排他的に2,3（その後、独自の内因性行列を生成するために進むことができる）は、細胞の構成unscaffoldedシステムとして懸濁液中で実施することができます。ここでは、心臓分化oをするためのプロトコルを提示しますFヒト多能性幹細胞（hPSCs - 身体の任意の細胞型になることができる幹細胞、胚または他のソースからも）サイズ制御、均一、unscaffolded凝集体です。 懸濁凝集体としてhPSCsの分化は、ラン内および実行間の両方の凝集サイズの大きな変動に悩まされています。この変動は、細胞コロニーを機械的に分離することを含む一般的に、これらの凝集体を生成するために使用される方法の結果です。この変動性を低減するために、多くのアプローチは、凝集体あたりの細胞数、並びに凝集体直径及び均一性を制御するために使用されています。吊り5をドロップとしては、例えば、マイクロ遠心チューブ中の凝集体の形成4、または、微細は、その後、UまたはV底マルチウェルプレート7,8に懸濁液、または細胞の遠心分離に転送することができる2次元HPSCコロニー6を定義しました9。しかし、これらすべてのAPPRoachesは集計世代の彼らの低いスループットによって制限されています。まあベースのシステムでは、V底プレートシステムに同様のアプローチ、マイクロウェルのしかし小さいサイズを採用するだけでなく、単一の培養プレートから均一な凝集体の多数の生成を可能にする（このプロトコルで各400ミクロンの幅を有します）全体のV底プレート10から生成されるよりも（〜1200マイクロウェルを含む〜15.5ミリメートルの標準直径）。まあベースの凝集体形成は、外胚葉から11 hPSCsの分化、内胚葉12、中胚葉13および胚体外14運命を含む多くの設定で使用されてきました。間葉系幹細胞15からの軟骨形成;毒物学的スクリーニング16に対して均一な基質の生成;そして、メカノ17の調査。 HPSC由来cardiomの製造のための強固な製造プロトコルを開発する上での一つの大きな課題yocytesは、実行間の心臓分化効率の再現性の欠如となっています。我々は以前、この変動が自己複製hPSCsと内胚葉および神経分化6,18に関連した遺伝子を発現する分化する細胞の両方を含む開始HPSC集団に異質に帰することができることを実証しました。これらの分化する細胞によって分泌された信号は、心臓の誘導に影響を与えます。神経前駆細胞は、心臓の誘導を阻害しながら、具体的には、胚体外内胚葉は、心臓の誘導を促進します。 HPSC集約の際、集約分化中の細胞と未分化hPSCsは、骨材表面13上で開発胚体外胚葉細胞の層に囲まれているように整理します。凝集体サイズを制御することによって、我々は、未分化hPSCs（体積比表面積）に対する心臓誘導内胚葉細胞の比率を調節し、最大心臓誘導の13のために、この比を最適化することができます。

<table border="0" cellpadding="0" cellspacing="0" width="645">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biological safety cabinet</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette aid</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">&#160;Serological pipettes (5 to 25 mL)&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aspirator</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aspirator or Pasteur pipettes</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;15 and 50 mL conical tubes</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fume hood</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.22 &#181;m syringe filter</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:216px;">5% CO2, 5% O2, and humidity controlled cell culture incubator&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td>Hypoxic (low oxygen) incubator</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:216px;">5% CO2, 20% O2, and humidity controlled cell culture incubator&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Low speed centrifuge with a swinging bucket rotor fitted with a plate holder&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">P2, P20, P200, and P1000 micropipettors and associated tips&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted microscope with 4X, 10X and 20X phase objectives&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra-Low Attachment (ULA) 24 well plates&#160;</td>
			<td style="width:143px;">Corning/Costar</td>
			<td style="width:119px;">3473</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.5 mL microcentrifuge tubes</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bench-top microcentrifuge</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Ascorbic Acid</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile Ultrapure distilled water</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:119px;">W3500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ice</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">-20C freezer</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Monothioglycerol</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td style="width:119px;">M6145</td>
			<td>Toxic; Aliquoting of MTG is strongly recommended to minimize oxidation due to repeated opening. Aliquots can be stored at 4 &#176;C for up to 3 months, -20 &#176;C is recommended for long-term storage.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">StemPro-34 Medium</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td>10639-011</td>
			<td>Basal Cardiac Induction Medium; The supplement is stored at -20 &#176;C and the basal medium at 4 &#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transferrin</td>
			<td style="width:143px;">Roche</td>
			<td>10652202001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">BMP-4</td>
			<td style="width:143px;">R&#38;D Technologies</td>
			<td style="width:119px;">314-BP</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">bFGF</td>
			<td style="width:143px;">R&#38;D Technologies</td>
			<td style="width:119px;">233-FB</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">VEGF</td>
			<td style="width:143px;">R&#38;D Technologies</td>
			<td style="width:119px;">293-VE</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Activin A</td>
			<td style="width:143px;">R&#38;D Technologies</td>
			<td style="width:119px;">338-AC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IWP-2</td>
			<td style="width:143px;">Reagents Direct</td>
			<td style="width:119px;">57-G89</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate buffered saline (PBS)</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14190</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15561</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrochloric acid&#160;</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td>258148</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethylsulfoxide (DMSO)</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DMEM/F-12</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12660</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100X Penicillin/Streptomycin</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100X L-glutamine</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25030</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Knockout Serum Replacement</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10828010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TrypLE Select</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12563</td>
			<td>Dissociation enzyme</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Aggrewell 400 plates</td>
			<td style="width:143px;">StemCell Technologies</td>
			<td style="width:119px;">27845</td>
			<td>Microwell Plates</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Aggrewell Rinsing Solution</td>
			<td style="width:143px;">StemCell Technologies</td>
			<td style="width:119px;">7010</td>
			<td>Microwell Rinsing Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Y-27632 ROCK Inhibitor</td>
			<td style="width:143px;">Tocris</td>
			<td style="width:119px;">1254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase Type II</td>
			<td style="width:143px;">Sigma-Aldrich</td>
			<td>C6885&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:143px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12483</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">96 well plate (for FACS staining)</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Intraprep Permeabilization Reagent</td>
			<td style="width:143px;">Beckman Coulter</td>
			<td>IM2389</td>
			<td>Kit with 2 parts: Fixation Solution and Permeabilization Solution; Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cTnT antibody</td>
			<td>Neomarkers&#160;</td>
			<td style="width:119px;">MS-295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat anti-mouse-IgG APC antibodyThermoFisher</td>
			<td style="width:143px;">Molecular Probes</td>
			<td style="width:119px;">A865</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5 mL round bottom flow cytometry tubes</td>
			<td style="width:143px;">FACS machine dependent</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

aggregate size optimization in microwells for suspension-based cardiac differentiation of human pluripotent stem cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

hPSCsのサイズ制御の凝集体を効果的にのみ細胞の濃度およびマイクロウェルの表面積に依存する、マイクロシステムを用いて形成することができます。短い遠心分離後、細胞（このプロトコルで千）の適切な番号が各マイクロウェル（ 図1A）にまとめられます。重要なことは、これらの細胞は、24時間以内に細胞内の接続を再確立し、もはや十分に埋めるべきではありませんが、滑らかなエッジ（ 図1B）とコンパクトな集合体として表示されます。これらの凝集体は、心臓の運命に向かってさらなる分化のための出発材料を提供します。細胞が密なクラスターを形成しない場合、これは、解離および再凝集次可能細胞死を示唆し、そして特定の細胞株のための単一細胞継代及びROCK阻害剤濃度の適合性を検討すべきです。マイクロウェルの次の3日間は、集計モルフォはほとんど変化を示していますでれっと、いくつかの成長は明らかですが。マイクロウェルから除去した場合、凝集体は、そのラウンド、密集した形態を維持し、互いに（ 図1C）と同様のサイズのものでなければなりません。 ULA 24ウェルプレートでの培養は、細胞増殖及び成長を可能にします。 8日目では、最初のアクチビンシグナリング、およびWnt阻害に暴露した後、凝集体は大きく、明るく、凝集体（ 図1D）として表示されるようになります。この期間中、かなりの細胞破片は、各ウェルの底に明らかであろうし、凝集体がメディアを削除する前に解決することを可能にすることによって除去されなければなりません。時折、多くの凝集体が互いに融合します。これらの「スーパー凝集体は「小さい凝集体および完全な分化を受ける可能性は低いと見られる形態学的変化を示さない傾向があるが、これは、十分に他の凝集体の分化を阻害しません。 大型化と組織化繊維状の領域の外観を持つ集合体に顕著な形態学的変化に= &quot;1&quot;&gt;続き分化結果-together.withinページを保つ：FO」jove_content」。日12で、収縮凝集を観察することができます。これらは常に大きな透明の細胞で構成され、多くの場合、骨材（ 図2）の外側に広範囲の細胞外マトリックスを含むことになります。集合全体の収縮が成功した分化を示し、一方、心臓マーカーの発現はまた、収縮していないようで凝集体で観察することができます。凝集体および免疫標識の解離に続いて、細胞の大部分は、フローサイトメトリーによる心筋マーカーのcTnT（ 図3）について陽性となります。このマーカーの発現は、細胞中で安定しておりとして後期分化の19日目のように、凝集体において観察されてもよいです。 1.JPG &quot;/&gt; 図1：HPSCのタイムラインは、心臓の運命に向かって分化した直後に集約した後、細胞はほぼ各マイクロウェル（A）を満たします。 1日後、凝集体が凝縮表示され、（B）を滑らかに。この形態は、凝集体がマイクロウェルから除去し、ウェルプレート（C）に播種した場合でも持続します。 8日目では、凝集体が拡大し始め、色（D）で明るく見えます。スケールバー：250μmで<a href="https://www.jove.com/files/ftp_upload/54308/54308fig1large.jpg" target="_blank">、この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/54308/54308fig2.jpg" /> 図2：凝集体は、トップパネル（強制的な集合体全体の収縮が観察された、心臓誘導ステージ3培地中で6日後に分化の12日目により締約開始 ：リラックスエーション、中央のパネル：収縮）。下のパネルは、黒または白（矢じり）として表示される最も重要な違いは、上部と中央のパネルを引いから誘導されます。スケールバー：250μmで<a href="https://www.jove.com/files/ftp_upload/54308/54308fig2large.jpg" target="_blank">、この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/54308/54308fig3.jpg" /> 図3：17日目差別hPSCsにおける心筋トロポニンTのための免疫標識 、細胞のほとんどは、フローサイトメトリー（充填されたヒストグラム）によって心筋トロポニンT陽性です。また、二次抗体単独（塗りつぶされていないヒストグラム）で染色した細胞が示されている。 <a href="https://www.jove.com/files/ftp_upload/54308/54308fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells at JoVE.com

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

ヒト多能性のサスペンション集計ベースの心臓分化を開始する従来の方法は、細胞（hPSCs）は凝集体の大きさや形状に関して培養異質で悩まされている茎。ここでは、サイズ制御HPSCは心臓の促進条件下で培養された凝集体を生成するためにマイクロウェルを用いた心臓分化のための堅牢な方法を説明します。

ヒト多能性幹細胞の懸濁液ベースの心臓分化のためのマイクロウェル中の凝集体サイズの最適化

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

aggregate-size-optimization-microwells-for-suspension-based-cardiac

Research

JoVE Journal

Developmental Biology

6.7K ビュー.  University of Toronto. ヒト多能性のサスペンション集計ベースの心臓分化を開始する従来の方法は、細胞（hPSCs）は凝集体の大きさや形状に関して培養異質で悩まされている茎。ここでは、サイズ制御HPSCは心臓の促進条件下で培養された凝集体を生成するためにマイクロウェルを用いた心臓分化のための堅牢な方法を説明します。 

ビデオ: Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

分化運命の空間組織を探るためのヒト多能性幹細胞のステンシルマイクロパターニング

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

発生生物学

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

再現性の高い小分子ベースの分化プロトコルを使用したヒト多能性幹細胞から心筋細胞の大規模生産

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

マイクロ電極アレイ上のネットワーク・アクティビティを測定するための人工多能性幹細胞の急速な神経分化

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

多能性幹細胞は、心筋の修復のための心臓細胞を派生します

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

ひと多能性幹細胞からプリミティブかつ決定的な造血前駆細胞の分化を監督

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

ヒト多能性幹細胞のin vitro分化における絨毛細胞へ

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

人間の胚または誘導多能性幹細胞からの網膜色素上皮細胞の急速な監督の分化

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

効率的かつスケーラブルな臨床的に互換性のある角膜輪部上皮幹細胞ひと多能性幹細胞からの分化

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

ヒト多能性幹細胞の肝細胞への効率的な分化

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

体節誘導体誘導される人間からの体外世代多能性幹細胞します。

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...