Work was funded by a Development and Promotion of Science and Technology scholarship from the Thai Ministry for Education to W.W. and grants from Tenovus and the Anonymous Trust to M.P.S.

1.メディアの準備注：プロトコルは、2つの別のメディアのミックスの使用に依存している：変更されたN2サプリメントを補充したDMEM / F12とするNeurobasalは、典型的には1に、B27サプリメントで補充：1の比率。 <ol><li> 15mlチューブに成分を混合することによって修正されたN2サプリメントを準備します。これをボルテックスしたりフィルタリングしません。溶液が透明になるまでチューブを転倒混和。 <ol><li> （0.01 M HClに作られた）25 mg / mlのインスリンの1ミリリットルを加え、チューブを反転させてよく混ぜ、その後、DMEM / F12の7.2ミリリットルをピペットで起動します。溶液が透明になるまでには数分かかります。 </li><li> 100ミリグラムの1ミリリットルを追加/ mlの（水の中に作られた）アポトランスフェリン、0.6ミリグラム/ 33μlのミリリットルprogesternone（エタノール中に作られた）、水に作られた160 mg / mlの（1 M）プトレシン（100μlの）、水の中に作られた3ミリモルの亜セレン酸ナトリウム（）10μlの7.5％ウシ血清アルブミンの666μlを、よくチューブを混ぜます。 -20℃で2.5ミリリットルに小分けとストア最大2ヶ月間C。 </li></ol></li><li>メディアを準備します。 <ol><li> DMEM / F12 250 mlにN2の2.5ミリリットルを追加します。よく混ぜますが振動やフィルタはありません。 </li><li>神経基礎の250ミリリットルにメディアはB27サプリメントの5ミリリットルを追加します。よく混ぜますが振動やフィルタはありません。 </li><li> 1：1の比のステップ1.2.1と1.2.2からメディアをミックス。いくつかのケースでは1：3混合物は、（：ステップ1.2.4で1ミックス1のように補足した）必要な場合があります。 </li><li>ミックスにL-グルタミン（最終濃度0.2 mM）の2-メルカプトエタノール（最終濃度0.1 mM）の3.5μlを0.5 mlのを追加し、振盪せずによく混ぜます。 3週間まで4℃でメディアを格納し、光にさらすことは避けてください。この最終的なミックスはN2B27と呼ばれています。 </li></ol></li><li>ゼラチン溶液を準備します。 <ol><li> 15分間、121℃、15 psiで超純水、オートクレーブ中の1％ゼラチン溶液を準備します。それは、このために使用ボトルは洗浄剤や消毒剤から完全にクリーンであることが重要なので、新しいボトルがあることをお勧めしますこのために使用されるとしか使用の間、超純水でリンスされます。 50ミリリットルのアリコートに1％溶液を分注し、数ヶ月のために4℃で維持。 </li><li>溶解するまで37℃の水浴中で1％ゼラチンのアリコートを温め、温かいリン酸緩衝食塩水（PBS）500ml中に加えます。十分に混合し、各ウェルまたはプレートの底をカバーするのに十分ピペット。少なくとも30分間コートにゼラチンを表面を許可します。 </li></ol></li></ol> 2.細胞をプレーティング注：このプロトコルは、白血病阻害因子（LIF）で10％血清中で増殖させたマウスESC、LIFおよび骨形成タンパク質4（BMP4）またはMEKとGSK3阻害剤（2Iメディア）とLIFまたは無血清培地と血清代替に等しく適用されますでまたはLIFなしで。しかし、分化のタイミングおよび効率は、細胞培地（説明を参照）に依存して変化し得ます。ここに示した実験のために、我々はEGFPレポーターがOにノックした46CマウスESC株を（使用しました10％血清およびLIFとGMEM中で増殖させた内因性SOX1対立遺伝子のNE）。最適な結果のためには、細胞が解離し、N2B27培地中に再プレーティングすることが重要です。単にGMEM /血清/ LIFからN2B27にメディアの変更は、常に細胞を再プレートと比較して減少分化効率をもたらします。 <ol><li> 10％血清、1mMピルビン酸ナトリウム、1×非必須アミノ酸、0.1 M 2-メルカプトエタノール、100ユニット/ mlのLIF 13 GMEM中で細胞を成長させます。 3日- 2かかりますT25培養フラスコにマウスESC、プレート1×10 6細胞のサブコンフルエント培養物を取得します。 </li><li>明視野顕微鏡で細胞を観察します。細胞がサブコンフルエントと通過する準備ができたら、PBS中で2回、それらをすすぎます。細胞解離試薬の1ミリリットルを追加し、2インキュベーターに戻す - 5分。トリプシンおよび他の酵素は、細胞分離のために用いることができるが、めっき密度があることが必要となるので、それらはadju細胞接着に悪影響を与えることができSTED。 </li><li>細胞がプレートから持ち上げるために始めているしたら、単一細胞懸濁液にそれらを取り除くために、容器をタップします。 15mlの遠心チューブに培地と細胞解離試薬およびピペットの10ミリリットルの合計に細胞を回収します。 </li><li> RTで3分間、300×gで細胞をスピン。 </li><li>慎重に上清を吸引し、ピペッティングにより3ダウン予め温めておいたN2B27 10mlにペレットを再懸濁 - 5回。ダウンサスをピペットすると、チューブの側面にピペットは、泡を作成しないように。自動細胞カウンターまたは血球計数器で細胞をカウントし、濃度を記録します。 </li><li>予め温めておいN2B27にウェルあたりするボリュームの単位当たりの細胞の所望の数を含む細胞懸濁液を準備します。 6ウェルディッシュ中で1cm 2当たり10,500細胞の密度（ すなわち、6ウェル皿のウェル当たり1×10 5個の細胞）が最適です。 cm 2であり、PLAあたりの細胞の推奨数については、表1を参照してください。細胞培養容器の様々なティンメディアボリューム。 </li><li>培養容器からゼラチンを吸引し、 表1で推奨密度とボリュームに応じて細胞懸濁液をプレート。これは中央に細胞を集中するようにプレートを旋回しないでください。 37℃、5％CO 2の湿潤インキュベーター内で培養を配置します。 </li><li>新鮮N2B27で全てのメディアを交換することによって、2日 - すべての1のメディアを変更します。ピペットは、細胞を穏やかにフラッシュなどの密度に影響を与え、次の日に分化効率を低下させる可能性があります。めっき後の初期の生存率が悪い場合には、1で細胞をプレート：N2及びB27添加培地の3ミックスと最初の2日後N2B27に変更。細胞が分化するように開始され、4の内部（ここで使用46C細胞株におけるレポーターの緑色蛍光によって可視）SOX1の発現を示すべきである- 6日。 </li></ol> 3.免疫蛍光染色 NOTE：プロトコルは、任意の容器型に適用することができます。各試薬の量は、6ウェルプレートのウェル毎に記載されており、任意の表面領域にスケーリングすることができます。再プレーティングは、その後低い生存能力にはお勧めしません。 <ol><li>分化培地を除去し、4％の500μL（v / v）のパラホルムアルデヒドを添加することにより、細胞を固定、15分間放置します。 </li><li>洗浄し、PBS-T（リン酸緩衝生理食塩水0.5％（v / v）のトゥイーン20）を2 mlの細胞透過性を2回。細胞を4℃で最長2ヶ月間、PBS-T中で維持することができます。 </li><li> 10％（二次抗体と同じ種に由来する）（v / v）の血清をPBS-T 1mlのPBS-Tを交換してください。任意の非特異的結合をブロックするために室温でロッカープレート上で1時間インキュベートします。 </li><li> 1時間後、PBS-Tを2 mlの細胞を2回洗浄し、（1希釈した10％の血清をPBS-Tで1000）、βIIIチューブリンに対する500μlのマウスIgGでインキュベートします。プレートロッカーに入れ、4℃でO / Nのままにしておきます。 </li><li>この段階から、常に血管を保護光から。次に（10％血清をPBS-T中の2μg/ mlに希釈した）蛍光標識抗マウスIgG抗体500μlを添加、PBS-Tで細胞を2回洗浄します。室温で1時間プレートロッカーの上に置きます。 </li><li>その後、DAPIの500μlを添加、PBS-Tで細胞を2回すすぎ（PBS-Tで0.5μg/ mlに希釈し）、5分間のままにします。 </li><li>再び細胞を洗浄し、PBS-Tに保管してください。細胞を撮影する準備ができていると、4℃で2週間まで維持することができます。それは固定され、生細胞の、又は異なる時間に固定された細胞のGFPの強度を比較することは不適切であるので、GFPシグナルが経時フェード注意。 </li></ol>

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The authors have nothing to disclose.

単層神経分化プロトコルは、十年6オーバーのために使用されてきました。プロトコルが定義された媒体で構成され、前臨床のためのシステムをより適用になり、単層システムで行われ、非常に効率的である（ 例えば、薬物スクリーニング）を使用しています。しかし、分化効率を決定するいくつかの重要な要因があります。この記事では、これらの要因と各障害物のためのソ...

胚性幹細胞（キメラを形成することによって）適切な段階の胚に再導入後のすべての成体細胞型に分化する能力を保持しながら、in vitroで無限に増殖する能力を有する初期胚から誘導された多能性細胞、同系または免疫無防備状態の宿主に注入されています適切な手がかりに（奇形腫を形成することにより）、またはインビトロ 、対象における 1。神経系統へのマウス胚性幹細胞のin vitroでの分化は、1995年に初めて記載され、モルフォゲン、レチノイン酸2-4を補った血清含有培地中の多細胞懸濁凝集体（胚様体に、EB）の形成を関与していました。それ以来、種々のプロトコルは、神経分化5を可能にするために開発されてきました。まだ凝集を利用する多くの、他の細胞型を誘導するとの共培養を、いくつかの無血清培地の使用を含みます。すべてのプロトコルの利点持っていますND欠点及び神経または神経細胞の正確な性質は、また、使用するプロトコルに応じて変化する生じました。 理想的なプロトコルは、堅牢で拡張性の高い、完全に定義された培地と基質を利用する、分化過程の非侵襲的モニタリングに適して、外部の手がかりによってパターニングすることができる神経前駆細胞の純粋な集団の生成をもたらすと区別するためであろう比較的短時間で高効率と収率で、すべてのニューロンとグリアサブタイプに。最後の十年では、低密度、無血清の接着性単層培養6-10にマウスESCから神経前駆細胞と神経細胞を生成するための方法を使用しています。このプロトコルは、上述した多くの基準を満たす：我々の手に分化効率は、長年にわたって非常に一貫しており、種々の細胞株は、スケールアップすることができ、またはダウン（我々が正常に96ウェルプレートから容器を用います直径15cmジシーズ）と使用するメディアは、明確に定義されています。プロセスは、（ドーパミン作動性ニューロン6のために、例えば、ShhのとFGF8）ニューロンのサブタイプの別個の種類の生成を誘導するために添加することができる分化のモニタリングおよびパターニング手がかりの様々なタイムラプス顕微鏡に適しています。 それにもかかわらず、このプロトコルの成功のアプリケーションにはいくつかの課題があります。重要な側面の一つは、メディアの入念な準備です。我々は常に商業選択肢の利用可能性にもかかわらず、社内メディアを準備します。使用サプリメントの一つ（N2; プロトコルを参照）は、標準的な市販のバージョンに比べて変更されています。最後に、この方法の成功した適用のために最も重要なステップの一つは、めっきに最適な細胞密度です。誘導信号（FGF4 11）のいずれかの自己分泌性質が最適viabilを可能にするのに十分な細胞が存在することを必要とするので、これは主に高すぎる密度分化における性と分化は、（おそらくLIF 12の自己分泌産生に起因する部分で）損なわれています。これは、培地調製および細胞のプレーティングの両方が最適な結果を保証するために慎重にかつ一貫して行われることが重要です。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11320-074</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Life Technologies</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro Accutase Cell Dissociation Reagent</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement, serum free</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I6634</td>
			<td>Reconstitute with sterile 0.01M HCl</td>
		</tr>
		<tr>
			<td>Apo-transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>Reconstitute with ethanol</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Sodium selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td>Reconstitute with sterile water</td>
		</tr>
		<tr>
			<td>Bovine albumin fraction V</td>
			<td>Life Technologies</td>
			<td>15260-037</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td>Make sure it is completely dissolved before use as glutamine is usually sedimented</td>
		</tr>
		<tr>
			<td>Gelatine</td>
			<td>Sigma</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture dish</td>
			<td>Thermo Scientific</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>GMEM</td>
			<td>Life Technologies</td>
			<td>11710-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acids solution</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr>
			<td>Leukaemia inhibitory factor</td>
			<td colspan="3">prepared in-house as in Smith 1991 Journal of Tissue Culture Methods</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>Mouse anti betaIII tubulin IgG antibody</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence-labelled anti-Mouse IgG antibody</td>
			<td>Life Technologies</td>
			<td>A31571</td>
			<td>Protect from light</td>
		</tr>
		<tr>
			<td>25cm2 tissue culture flask</td>
			<td>Thermo Scientific</td>
			<td>156367</td>
			<td></td>
		</tr>
	</tbody>
</table>

neural differentiation of mouse embryonic stem cells in serum-free monolayer culture

神経前駆細胞にマウス胚性幹細胞（ESC）の分化する能力は、神経の仕様を制御する機構の研究だけでなく、さらなる研究のために成熟した神経細胞型の生成を可能にします。このプロトコルでは、無血清、単層培養を用いて、神経前駆細胞へのESCの分化のための方法を説明します。この方法は効率的で、拡張可能であり、4内の70％の神経前駆細胞〜の産生をもたらす - 6日。これは、様々な条件下で成長させた様々な株からESCに適用することができます。神経前駆細胞は、機能性ニューロンおよびグリアへとさらに分化させ、または顕微鏡検査によって分析し、フローサイトメトリーまたは分子技術を流すことができます。分化過程をタイムラプス顕微鏡検査に適していると神経仕様プロセスを監視するためのレポーター系の使用と組み合わせることができます。私たちはBに処理を可能にするために、メディアの準備と細胞密度の最適化に関する詳細な指示を与えますEは最もESC株および細胞培養容器の様々な適用しました。

この実験では、神経分化を追跡するために、内因性SOX1-GFPレポーターと46C細胞ライン14、マウス胚性幹細胞を使用します。この細胞株、SOX1の発現は、神経前駆細胞のマーカーを使用することにより、緑色蛍光により検出することができます。密度をメッキすることは、神経分化を達成するための重要な要因です。マウス胚性幹細胞は、10,500 88500個の細胞/ cm 2で変化する異なる?...

Watch this Scientific Journal Video about Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture at JoVE.com

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

無血清単層培養中のマウス胚性幹細胞の神経分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

neural-differentiation-mouse-embryonic-stem-cells-serum-free

Research

JoVE Journal

Developmental Biology

14.0K ビュー.  University of Dundee. This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

ビデオ: Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Basicおよび前臨床研究のための密閉型細胞培養系でヒト多能性および神経幹細胞を培養します

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

発生生物学

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

胚性幹細胞や組織の心臓分化のエピジェネティック制御

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

創薬に適していEMT調査のための酵素および無血清神経幹細胞培養モデル

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

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 ...

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 ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for studying early embryonic development. In the absence of leukemia inhibitory factor (LIF), ESCs differentiate by default into neural precursor cells. They can be amassed into a three dimensional (3D) spherical aggregate termed embryoid body (EB) due to its similarity to the early stage embryo. EBs can be seeded on fibronectin-coated coverslips, where they expand by growing two dimensional (2D) extensions, or implanted in 3D collagen matrices where they continue growing as spheroids, and differentiate into the three germ layers: endodermal, mesodermal, and ectodermal. The 3D collagen culture mimics the in vivo environment more closely than the 2D EBs. The 2D EB culture facilitates analysis by immunofluorescence and immunoblotting to track differentiation. We have developed a two-step neural differentiation protocol. In the first step, EBs are generated by the hanging-drop technique, and, simultaneously, are induced to differentiate by exposure to retinoic acid (RA). In the second step, neural differentiation proceeds in a 2D or 3D format in the absence of RA.

We describe a technique for using murine embryonic stem cells for generating two or three dimensional embryoid bodies. We then explain how to induce neural differentiation of the embryoid body cells by retinoic acid, and how to analyze their state of differentiation by progenitor cell marker immunofluorescence and immunoblotting.

2および3次元の胚様体中のマウス胚性幹細胞のレチノイン酸誘発神経分化の分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

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 ...

Generation of Induced Neural Stem Cells from Peripheral Mononuclear Cells and Differentiation Toward Dopaminergic Neuron Precursors for Transplantation Studies

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral mesencephalon (VM). Cell replacement therapy holds great promise for treatment of PD. Recently, induced neural stem cells (iNSCs) have emerged as a potential candidate for cell replacement therapy due to the reduced risk of tumor formation and the plasticity to give rise to region-specific neurons and glia. iNSCs can be reprogrammed from autologous somatic cellular sources, such as fibroblasts, peripheral blood mononuclear cells (PBMNCs) and various other types of cells. Compared with other types of somatic cells, PBMNCs are an appealing starter cell type because of the ease to access and expand in culture. Sendai virus (SeV), an RNA non-integrative virus, encoding reprogramming factors including human OCT3/4, SOX2, KLF4 and c-MYC, has a negative-sense, single-stranded, non-segmented genome that does not integrate into host genome, but only replicates in the cytoplasm of infected cells, offering an efficient and safe vehicle for reprogramming. In this study, we describe a protocol in which iNSCs are obtained by reprogramming PBMNCs, and differentiated into specialized VM DA neurons by a two-stage method. Then DA precursors are transplanted into unilaterally 6-hyroxydopamine (6-OHDA)-lesioned PD mouse models to evaluate the safety and efficacy for treatment of PD. This method provides a platform to investigate the functions and therapeutic effects of patient-specific DA neural cells in vitro and in vivo.

The protocol presents the reprogramming of peripheral blood mononuclear cells to induce neural stem cells by Sendai virus infection, differentiation of iNSCs into dopaminergic neurons, transplantation of DA precursors into the unilaterally-lesioned Parkinson&#39;s disease mouse models, and evaluation of the safety and efficacy of iNSC-derived DA precursors for PD treatment.

末梢単核細胞からの誘導神経幹細胞の生成と移植研究におけるドーパミン作動性ニューロン前駆体への分化

Parkinson&#39;s disease (PD) is caused by degeneration of dopaminergic (DA) neurons at the substantia nigra pars compacta (SNpc) in the ventral ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

多能性幹細胞からの3次元ヒト肝球の血清自由産生

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

ヒト胚性幹細胞の単細胞培養を用いた効率的な神経分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...