この作品は、部分的に国立衛生研究所（RO1 NS059043とRO1 ES015988）、全国多発性硬化症協会、貧血の研究、フェルドスタイン医学財団のためのロシュ財団、およびシュライナーズ子ども病院からWDへの補助金によって支えられている。 我々は提案のためにデビッドの喜びとジェニファープレーンを感謝したいと思います。我々はこの記事に関連する一切の競合する利害を宣言しません。

GFP - Olig2マウス胚性幹細胞（mESCs）からオリゴデンドロサイト前駆細胞（OPCを）生成の詳細な手順： <ol><li>マウスES細胞株GFP - Olig2（G - Olig2）は、アメリカンタイプカルチャーコレクション（ATCC）から購入しています。 mESCsは、日常的に照射したマウス胚性線維芽細胞（MEF）フィーダー層の上に3日毎に継代されています。 MEF細胞はES細胞を継代する前に少なくとも一日0.1％ゼラチンコートした6ウェル組織培養プレートに播種されています。 MESC培地は、20％ウシ胎児血清（GIBCO）、2mM L -グルタミン、1mMピルビン酸ナトリウム、0.1mMのβ-メルカプトエタノール/、1％非必須アミノ酸（を添加したダルベッコ改変イーグル培地（DMEM）（GIBCO）です。 NEAA）、および1,000 U / mlの白血病抑制因子。 </li><li> MESのコロニーは単一の細胞に（TrypLE、5分、37 ° C）をトリプシン処理し、ノックアウト血清代替（KSR）培地に懸濁されて、その後、細胞は細胞でコスター超低アタッチメント6ウェルプレート（コーニング）に転送されます。 50,000細胞/ cm 2の密度。これらの条件では、mESCsは胚様体（EB）を形成する。 KSRの培地は、20％KSR、1mMピルビン酸ナトリウム、1％NEAA、および0.1mMβ-メルカプトエタノール/を補充必須培地（- MEM） - 最小限で構成されています。 </li><li> 4日から7日目に、レチノイン酸（RA、0.2 M）とpurmorphamine（ピュール、1 M）は、図2に示すように含まれています。 KSRまたはN2培地中の1A。 N2の媒体は1X N2サプリメント、1mMピルビン酸ナトリウム、1％NEAA、および0.1mMβ-メルカプトエタノール/を含む- MEMです。培地は毎日変更されます。 </li><li> 8日目で、EBはN2培地および線維芽細胞増殖因子-2（FGF - 2、20 ng / ml）のから構成されるOPCの培地中0.01％ポリオルニチンコートしたディッシュ上TrypLE（5分、37℃）およびメッキを使用して細分化されています。培地は2日毎に変更されます。 </li><li>細胞はトリプシン処理（TrypLE、3分、37 ° C）と彼らがコンフルエントになるとほぼ毎週色を保ち続けるされています。 30日目では、細胞の80％以上がGFP/Olig2表現を見せると、NG2陽性OPCの特徴です。 </li><li>スパイクOPCを生成するには、BacMamのNa +チャネルのキットは、これらのMESC由来のOPCはにのNa V 1.2サブユニットを導入するために使用されます。 BacMam試薬（1 mlの成分）5 mlの最終量になるようにPBSと混合される。その後、混合BacMam試薬はPBSで洗浄する前に60 mmの培養皿（50〜70％の合流点）でMESC由来のOPCは文化に追加されます。 2時間のインキュベーションの後、混合BacMam試薬は1Xエンハンサーを添加したOPCの培地を除去し置き換えられます。エンハンサーは、BはDMSO（成分C）中で再構成するコンポーネントです。次に、追加の2時間のインキュベーションの後にエンハンサーを持つ媒体は完全なOPCの培地を除去し置き換えられます。細胞を24時間後にアッセイされる。 </li></ol>代表的な結果： 図に示すように差別化プロトコルに従って。 1A、G - Olig2のmESCsは、最初に胚様体（EB）（図1B）を形成するためにKSR培地で4日間懸濁した。その後、私たちは順番にRAとピュールでEBSを処理した。 EBは、4日目で、任意のGFPの蛍光を示し、D8（図1B）で強いGFPの蛍光を発現していない。この時点で、EBは成長因子FGF - 2を添加したN2培地でトリプシン処理し、播種した。 22日（D30）さらに培養後、GFP +細胞の割合は80.3に達し、± 0.6％を私たちのフローサイトメトリーの結果によると。図に示す。 1C、GFP +細胞は一貫してNG2染色（96.4 ± GFP陽性細胞の1.3％プラスのNG2、および94.8 ± NG2陽性細胞の1.5％がGFP陽性であった）と重なる。従って、これらの細胞は、OPCとして定義する、GFP/Olig2とNG2を表現。 MESC由来のOPCは（76セル）脱分極（図2A）により活動電位を発生させることに失敗しました。のNa V 1.2サブユニットを運ぶバキュロウイルスで形質導入した後、これらのMESC由来のOPCは（17細胞の94.1％、16）（図2B）スパイクに分類される。 <img alt="図1" src="/files/ftp_upload/1960/1960fig1.jpg" /> 図1。 OPCはにGOlig - MESCの分化。胚様体（EB）をベースと小分子駆動型分化のプロトコルを示す（）スキーム。 D8で、EBは、細分化し、メッキだった。彼らがコンフルエントになった時点で細胞は1週間に1回継代した。 （B）D8ではなく、D4 EBは、GFPの発現を示した。 （C）NG2（赤）の免疫染色では、D30でのGFP（緑色）の発現と一致していた。 DAPI（青）は核を識別するために使用されました。スケールバー：20 mmです。 <img alt="図2" src="/files/ftp_upload/1960/1960fig2.jpg" /> 図2。でMESC由来のOPCをnonspikingからスパイクOPCの世代のウイルス媒介セルが0に脱分極した場合でも、 ナトリウムVチャネルの発現。（A）膜電位が-60 mVとMESC由来のOPCが開催されましたが、活動電位を発生させることに失敗しましたmVの。 （B）MESC由来のOPCの例ウイルスの導入後の活動電位を（赤で強調表示）焼成。

<ol>
	<li>Xian, H., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dividing+Olig2-expressing+progenitor+cells+derived+from+ES+cells.">Dividing Olig2-expressing progenitor cells derived from ES cells.</a> Glia. 47 (1), 88-101 (2004).</li><li>Clair, A., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subset+of+ES-cell-derived+neural+cells+marked+by+gene+targeting.">A subset of ES-cell-derived neural cells marked by gene targeting.</a> Stem Cells. 21 (1), 41-49 (2003).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154-156 (1981).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Shin, S., Xue, H., Mattson, M. P., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-dependent+Olig2+expression+in+motor+neurons+and+oligodendrocytes+differentiated+from+embryonic+stem+cells.">Stage-dependent Olig2 expression in motor neurons and oligodendrocytes differentiated from embryonic stem cells.</a> Stem Cells Dev. 16, 131-141 (2007).</li><li>Billon, N., Jolicoeur, C., Ying, Q. L., Smith, A., Raff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+timing+of+oligodendrocyte+development+from+genetically+engineered,+lineage-selectable+mouse+ES+cells.">Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells.</a> J Cell Sci. 115, 3657-3665 (2002).</li><li>Nishiyama, A., Lin, X. H., Giese, N., Heldin, C. H., Stallcup, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-localization+of+NG2+proteoglycan+and+PDGF+alpha-receptor+on+O2A+progenitor+cells+in+the+developing+rat+brain.">Co-localization of NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells in the developing rat brain.</a> J Neurosci Res. 43, 299-314 (1996).</li><li>Ligon, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+NG2+neural+progenitor+cells+requires+Olig+gene+function.">Development of NG2 neural progenitor cells requires Olig gene function.</a> Proc Natl Acad Sci U S A. 103, 7853-7858 (2006).</li><li>Dawson, M. R., Polito, A., Levine, J. M., Reynolds, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-expressing+glial+progenitor+cells:+an+abundant+and+widespread+population+of+cycling+cells+in+the+adult+rat+CNS.">NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS.</a> Mol Cell Neurosci. 24, 476-488 (2003).</li><li>Chittajallu, R., Aguirre, A., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-positive+cells+in+the+mouse+white+and+grey+matter+display+distinct+physiological+properties.">NG2-positive cells in the mouse white and grey matter display distinct physiological properties.</a> J Physiol. 561, 109-122 (2004).</li><li>Karadottir, R., Hamilton, N. B., Bakiri, Y., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiking+and+nonspiking+classes+of+oligodendrocyte+precursor+glia+in+CNS+white+matter.">Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter.</a> Nat Neurosci. 11, 450-456 (2008).</li></ol>

利害の衝突は宣言されません。

胚盤胞の胚から単離した胚性幹細胞（ESC）は、オリゴデンドロサイトの仕様を含め、初期の哺乳類の発達を研究するためのin vitroのモデル系の提供、生物3、4のすべての細胞系譜に分化することができる。 mESCsは、ソニックヘッジホッグ（Shh）5の治療法とオリゴデンドロサイト前駆細胞体（OPC）に分化することが示されている。また、mESCsからShhの誘発性OPCの分化は、胚発生6で観察された正しいタイミングを維持します。したがって、mESCsからin vitroでのOPC 分化の性質は、in vivoでの開発でから学んだされているものと一致するように考えられている。ここでは、小分子RAとShhのアゴニストピュールを使用して、我々は成功し、高効率でGFP + Olig2 + NG2 + OPCはにG - Olig2 mESCsを分化した。 プロテオグリカンNG2 7とヘリックス-ループ-ヘリックス転写因子Olig2 8の発現によって特徴付けOPCは、、彼らは全細胞のかなりの割合（〜5％）を構成している発展と成熟した中枢神経系におけるオリゴデンドロサイトを、生成する主な増殖細胞のタイプ9。研究者はVチャネル Naは実証しているほぼ二十年のためのOPCの亜集団に発現し、脱分極10,11によって起動することができます。また、最近の印象的な観察​​9は その場ラットの中枢神経系の白質、OPCは（〜50％）生成された活動電位の電位依存性ナトリウム（Na V）チャネルの発現に応じ時に脱分極することであり、したがって、スパイクに細分することができますと亜集団をnonspiking。しかし、これらのスパイクの特性の関数はまだほとんど知られていない。我々は、電気生理学的に、Shhの依存プロトコルで差別化MESC由来のOPCは、 その場脳のOPCの場合と同じではないことがわかった。サブユニットのNa V 1.2を導入した後、これらのサイレントMESC由来のOPCはスパイクのことができた。 従って、MESC由来のOPCは、ここで説明分化プロトコルが容易に可能スパイク/ nonspiking（1）OPCをスパイクとnonspikingの機能の違いを勉強し、（2）スクリーニングMESC由来のOPCのでナトリウムチャネルの発現を促進することができる新しい要因、（ 3）開発と人間のESCまたは誘導多能性幹細胞（性IPSC）からOPCの分化のプロトコルを最適化。

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		<th>Material Name</th>
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<td>MEFs</td>
<td>&nbsp;</td>
<td>GlobalStem </td>
<td>GSC-6001G</td>
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<td>TrypLE Express</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12604</td>
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<td>Retinoic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R-2625</td>
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<td>Purmorphamine</td>
<td>&nbsp;</td>
<td>Cayman Chemical</td>
<td>10009634</td>
<td>&nbsp;</td>
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<td>FGF-2</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>GF003</td>
<td>&nbsp;</td>
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<td>BacMam NaV 1.2</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>B10341</td>
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differentiation of embryonic stem cells into oligodendrocyte precursors

オリゴデンドロサイトは、中枢神経系のmyelinating細胞である。脱髄疾患における再生細胞療法の場合は、移植のための系統にコミットされたオリゴデンドロサイト前駆細胞体（OPC）の純粋な集団を導出に大きな関心が寄せられている。 OPCは、転写因子Olig2とプロテオグリカンNG2の表面発現の活動によって特徴付けられる。 GFP - Olig2（G - Olig2）マウス胚性幹細胞（MESC）レポーターのラインを使用して、我々はGFP + Olig2 + NG2 + OPCはにmESCsの分化のための条件を最適化。我々のプロトコルでは、まずmESCsから胚様体の世代（EBS）を説明します。オリゴデンドログリア系譜に直接EBの分化に定義された培養条件下で（1）レチノイン酸（RA）及び（2）ソニックヘッジホッグ（Shh）アゴニストpurmorphamine（ピュール）：第二に、我々は、小分子とMESC由来のEBの治療について説明します。このアプローチによって、OPCは、30日の期間に高効率（&gt; 80％）で得ることができます。このプロトコルでmESCsに由来する細胞は、一次組織培養から派生したOPCのに表現型が類似しています。 MESC由来のOPCはその場で脳のOPCのサブポピュレーションのために説明するスパイクプロパティが表示されません。この電気生理学的性質を調べるために、我々は、異所的にNaの発現させることにより、MESC由来のOPCをスパイクの発生を説明<sub&gt; V</sub&gt; 1.2サブユニット。このプロトコルから得られたスパイクとnonspiking細胞は、OPCの二つ亜集団での機能研究を進めるのに役立ちます。

Watch this Scientific Journal Video about Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors at JoVE.com

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

我々は、小分子ベースのオリゴデンドロサイト前駆細胞体（OPC）にマウス胚性幹細胞の分化のためのプロトコルについて説明します。このプロトコルは、分化の30日間で高効率とOlig2 + NG2 + OPCを生成します。我々はまた、活動電位を発生させることができる&quot;スパイク&quot;OPCを生成する方法を説明します。

オリゴデンドロサイト前駆体に胚性幹細胞の分化

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

differentiation-embryonic-stem-cells-into-oligodendrocyte

Research

JoVE Journal

Biology

15.2K ビュー.  School of Medicine, University of California, Davis. 我々は、小分子ベースのオリゴデンドロサイト前駆細胞体（OPC）にマウス胚性幹細胞の分化のためのプロトコルについて説明します。このプロトコルは、分化の30日間で高効率とOlig2 + NG2 + OPCを生成します。我々はまた、活動電位を発生させることができる"スパイク"OPCを生成する方法を説明します。

ビデオ: Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

ヒト胚性幹（ES）細胞の増殖

生物学

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

マウス神経幹細胞の前駆体の文化

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

マウス胚性幹細胞から造血幹細胞の導出

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

免疫手術でヒト胚性幹細胞の導出

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

ヒト胚性幹細胞からクロマチン免疫沈降

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

ハンギングドロップ法を用いたマウス胚性幹（MES）細胞のin vitroにおける分化

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

マウスES胚細胞の単離と導出

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

運動ニューロンにマウス胚性幹細胞の効率的な分化

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

生殖様細胞に新生マウス皮膚由来幹細胞の分化<em&gt;インビトロ</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...