著者は、元のプロトコルのためにジャネットRossantに感謝。本研究では、WHの保健助成金CA106308の国立研究所によってサポートされていました。

このプロトコルでは、我々はマウス胚盤胞とTS細胞の樹立の準備について説明します。自然交配と超排卵の誘導のために設定を含む事前の胚盤胞のコレクションへの一般的なマウス操作では、、基本的にはナギらによって示される標準のプロトコルに従ってください。2003（pp146 - 150）。 TS細胞の派生とメンテナンス<ol><li>興味のマウスとの間のセットアップの繁殖クロス。 </li><li>フィーダー細胞としてマウス胚性繊維芽細胞（MEF）を準備する。二日胚盤胞の集合前に、MEFのは（2 × 10 6細胞）は、100 mmディッシュに播種されています。翌日は、これらの細胞は37℃で2時間のインキュベーションのためにマイトマイシンC（20 mg / mL）を含む° C、5％CO 2の TSの培地10mlで処理されています。これは、PBSで細胞を2回洗浄することによって続いている。マイトマイシンC処理した細胞を12ウェルプレート（1 × 10 5細胞/ウェル）に播種されています。 </li><li>胚盤胞のコレクション（3.5 DPC、初日と呼ばれる）の日に、MEFの培地は、TS培地に加えて倍速FGF4/Heparin液（0.5ml /ウェル）に置き換えられます。 </li><li>子宮のコレクションのための準備：マウスを安楽死させると子宮（図1A）を探します。 </li><li>子宮頸部（膀胱の後ろにある）で、ピンセットでそれを把握し、子宮を取り出して、子宮と子宮頸部（図1B）の接合間にカット。子宮を引っ張って、膜と脂肪組織を切り落とすと卵管との接合（図1C）下子宮をカット。 60 mmのプレート内のTS培地の少量（0.2 ml）に子宮（図1D）を配置します。 </li><li> 26ゲージの針で1mlのシリンジを使用してTS培地1mlでそれぞれの子宮の角をフラッシュします。子宮の底に針を挿入し、1 mlのTS培地で新しい60 mmのプレートにゆっくりと洗い流してください。子宮が膨張し、フラッシング中に完全に拡張する必要があります。 </li><li>口制御ピペットを使用してTSの培地を含むクリーンな12ウェルプレートに慎重胚盤胞を収集して転送する。 12ウェルプレートでTS媒体を介してシリアル転送で胚盤胞を3回洗浄する。 37マイトマイシンC処理MEFフィーダーや文化を含む° C、5％CO 2を 12ウェルプレートにウェル当たり片足胚盤胞（図2A）。 </li><li>胚盤胞は透明帯から孵化し、24〜36時間でウェルにアタッチされます。小さな突起は容易に3日目に観察される。 500μlの新鮮なTSの培地に加え、1 × FGF4/Heparinと文化を養う。 </li><li> 4または5日目に、胚盤胞の伸長は、そのサイズに応じて分解するための準備ができているはずです。 TSの細胞解離のための理想的なサイズは、図3Bに示されています。 0.5mlのPBSで一回の文化を洗ってください。 37℃で5分間、100ミリリットル0.25％トリプシン/ EDTAとインキュベートする° C、5％CO 2を 。 P100のpipettemanと激しく上下にピペッティングして小さな塊に伸長を破る。井戸に（30％のTS培地、70％MEF - CMプラス1.5 FGF4/Heparin）TS - 70CM - 1.5F4Hを追加することによって、トリプシン処理を停止します。 8時間分解した後、培地を変更し、2日毎に細胞を供給し続けます。 </li><li>日間で6と10（高度に可変）TS細胞のコロニーを観察することができます。彼らは明確なコロニーの境界（図2C）との形態のような平らな、上皮シートを持っている。 2日毎に培養液を供給し続ける</li><li> TS細胞がコンフルエント50％（通常は日15〜20の間）に到達すると、PBSで1回洗浄し、100ml 0.25％トリプシン/ EDTAを加える。近い単一細胞懸濁液を確実にするためにトリプシン処理中に上下にピペッティング。 TS - 70CM - 1.5F4HとマイトマイシンC処理したMEFフィーダー（2.5 × 10 5 cells/6-wellまたは5 × 10 5 cells/60mmプレート）を含む新たな6穴または60ミリメートルプレートへの転送を追加することにより、トリプシン処理を停止します。 </li><li> 8時間経過後に培地を変更し、2日毎に培養液を供給し続けます。 </li><li> MEFフィーダー上に1つまたは2つ以上の通路（通路の間に3〜5日）した後、TSの細胞はそれらなしで培養することができる。 MEFのは、はるかに高速にトリプシン処理後のTS細胞より培養皿に付着するので、接着速度でそのような違いは、MEFフィーダーからTS細胞を分離するために利用することができます。培養細胞を通過した後、° C、5％CO 2で30分間37℃、それらをインキュベートする。ほとんどのMEFはプレートに付着し、TSの人口は、懸濁液中に残ります。新しいプレートに上清を移す。純粋なTSの人口を取得するには、いくつかの文章について、この手順を繰り返します。 </li><li> TS - 70CM - 1.5F4HにTS細胞を維持し、4日ごとに渡す（1:10〜1:20の分割）や、文化が70％コンフルエントに達する。高密度（1:5分割よりも高い）または培養することを過度に合流、その分化を引き起こす可能性のある細胞を渡す。 </li><li> TS細胞の同一性は、細胞マーカー、Cdx2の（図3）の免疫染色によって確認することができます。 </li></ol>凍結TS細胞<ol><li>凍結培地（50％FBS、30％のTS培地および20％DMSO）を2倍用意してください。 </li><li>共同トリプシン処理によってllectのTS細胞は、6分の場合は450gで遠心分離した。 2倍速凍結培地の等量でTS培地に再懸濁します。 </li><li>徐々に-70℃でイソプロピルアルコールチャンバーを用いて細胞を凍結℃、24〜48時間後に液体窒素への転送。 </li><li> 60 mmのプレート（コンフルエント70％）通常2バイアルに保存されています。 </li></ol> TS細胞を融解<ol><li>すぐに37バイアルを解凍° Cは、（450グラム、6分）遠心して10ミリリットルTSのメディアでそれらを収集し、そして30％TSメディア、70CM - 1.5F4Hに再懸濁します。 </li><li>細胞の1バイアルを60 mmのプレートに播種されています。 </li></ol> TGCにTS細胞の分化栄養膜巨細胞（TGCs）にTS細胞の分化は、MEF - CM、FGF4とヘパリンの撤退により誘導され得る。 TGC表現型は（大核）は4日間の誘導後に検出することができる（田中ら、1998とチウら 2008）。 p450sccの免疫染色は、差別化された文化の中でこのTGCマーカー（図4A）の発現を検出する。 PI（ヨウ化プロピジウム）染色された細胞のフローサイトメトリー分析は、さらに高いDNA含量（図4B）を含む分化した細胞の存在を示しています。これらの結果は、TS細胞が倍数体TGCsになるために核内倍加を受けることを示唆している。 代表的な結果<img src="/files/ftp_upload/1964/1964fig1.jpg" alt="図1" /> 図1マウスの子宮の解剖。 （A）子宮の位置は矢印で示されます。に示すように（B）子宮および子宮頸部の接合間にカット。 （C）だけで卵巣（OV）の下に卵管（外径）以下の子宮をカット。子宮の（D）イメージ。 <img src="/files/ftp_upload/1964/1964fig2.jpg" alt="図2" /> 図2代表的な写真は、マウスの胚盤胞、胚盤胞の伸長や栄養膜のコロニーを示しています。 （A）E3.5で収集されたマウスの胚盤胞が表示されます。 ICM、内部細胞塊、MT、壁画の栄養外胚葉、PT、極性の栄養外胚葉。 （B）MEFフィーダー上に栄養膜伸長が表示されます。 TS、栄養膜幹細胞。 （C）栄養膜幹細胞のコロニーは、MEFフィーダー上に明確なエッジを示しています。矢印は、セル境界を示している。 <img src="/files/ftp_upload/1964/1964fig3.jpg" alt="図3" /> 図3解析を免疫染色によりTS細胞の同定。 TS（AD）とES（制御、EH）の細胞は、マーカーの同定の対象となります。細胞は、Oct4の（A、E）またはCdx2の（B、F）を認識する抗体で免疫染色し、マージされた画像で提示DAPI（C、G）（D、H）によって対比されています。スケールバーは50μm。 <img src="/files/ftp_upload/1964/1964fig4.jpg" alt="図4" /> 図4 in vitroでのTS細胞の分化。 （A）未分化（Undiff）または差別化（差分）細胞培養においては、TGCのマーカーの発現について調べたところ、p450scc（緑）、DAPI（赤）で対比。 （B）PIで染色された分化した細胞のフローサイトメトリー分析は、DNAの内容を（; M2、以上の4つのコピーM1、2〜4枚）を測定します。 M2の人口は、倍数体栄養膜細胞を表しています。

<ol>
	<li>Chiu, S. Y., Asai, N., Costantini, F. &. a. m. p. ;. a. m. p., Hsu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-specific+protease+2+is+essential+for+modulating+p53-Mdm2+in+development+of+trophoblast+stem+cell+niches+and+lineages.">SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages.</a> PLoS Biol. 6, E310-E310 (2008).</li><li>Kunath, T., Arnaud, D., Uy, D. G., Okamoto, I., Chureau, C., Yamanaka, Y., Heard, E., Gardner, R. L., Avner, P., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinted+X-inactivation+in+extra-embryonic+endoderm+cell+lines+from+mouse+blastocysts.">Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.</a> Development. 132, 1649-1661 (2005).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: The Laboratory Manual. , (2003).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

利害の衝突は宣言されません。

このビデオでは、我々は、TS細胞株を確立するために子宮や実験手順からE3.5胚盤胞を収集するプロセスを示しています。我々はまた、TS細胞のstemnessを維持し、分化した細胞への分化を誘導する条件について説明します。純粋なTS細胞株を取得する2つの重要なステップは、伸長の分解のためのタイミング（手順9）と初通過（ステップ11）処理です。それは、大規模な胚盤胞は、手順9または手?...

試薬 TS媒体： RPMI - 1640、20％FBS、1mMピルビン酸ナトリウム、100mMのβ-メルカプトエタノール、100 mg / mlのペニシリン - ストレプトマイシン。 マイトマイシンC： 2ミリリットルのPBSで2 mgをマイトマイシンC（シグマM - 0503）を溶解し、200ミリリットルのTS培地（10 ng / ml）の中にこの2mlの混合物を追加。使用するまで-20℃で20分注し（10ml）とストアを行います。 100 mmのプレートごとに一定量を追加。 マウス胚性線維芽細胞条件培地（MEF - CM）： 100 mmのプレート（1 × 10 6 /プレート）にプレートのMEF。翌日、皿当たり10mlのTS培地を追加し、48時間培養を続ける。それらをフィルタリング（0.45 mm）とを35mlのアリコートで-20℃で保存し、培養液を集める。 4℃で保存することができます必要に応じて各アリコート℃を解凍MEFのは、彼らがコンフルエントになる前に、CMの2つのバッチを準備するために使用することができます。 PBS / BSA： 0.45ミリメートルシリンジフィルターを通してフィルターPBS（10ml）に、のBSA、フラクションV（シグマA3311、0.1％（w / v）の）溶解し、1.5 mlチューブに1 mlのアリコートを行います。 -70℃で保存し、FGF4を準備するために必要な雪解けつのチューブを。 FGF4（1000X）： PBS / BSA溶液1 mlで再懸濁し凍結乾燥FGF4（シグマF8424、25mg）を。 -70℃でよく混ぜ合わせ、1.5 mlチューブ用と店舗に10アリコート（100ml）を加える℃に必要に応じて各チューブを融解し、4で、残りを保存° Cは、再凍結しないでください。 1X FGF4（25 ng / ml）を取得するためにTS培地で1000倍に希釈する。 ヘパリン（1000倍）。 1 mg / mLの（1000倍）の最終濃度にPBSで再懸濁し、ヘパリン（シグマH3149、1万台）。 -70℃で1.5 mlチューブや店舗に100 mlのアリコートを作る℃に雪解けのアリコートに必要と4で、残りを保存° Cは、再凍結しないでください。 1Xヘパリン（は1 ng / ml）を得るために、TS培地中に1000時間を希釈する。

derivation of mouse trophoblast stem cells from blastocysts

栄養外胚葉の仕様は、哺乳類の発生の最も初期の分化のイベントの一つです。栄養外胚葉を仲介移植から派生し、絨毛の系統は、胎盤の胎児の一部を生成します。その結果、この系統の開発は、胚の生存に必須です。マウスの胚盤胞から栄養膜幹細胞（TS細胞）の導出は、最初に田中によって記述されていた<em&gt;ら。</em&gt; 1998。適切な刺激の後のステージと細胞タイプ特異的マーカーのトロホブラスト特定のプロパティとそれらの発現を維持するためにTS細胞の能力は、初期の胎盤形成のイベントをrecapitulatingにより栄養膜の系統の開発を調査する貴重なモデルシステムを提供します。さらに、栄養膜細胞は、天然のゲノム増幅を受けているいくつかの体細胞のタイプのいずれかです。栄養膜polyploidizationの根底にある分子経路が解明し始めているものの、栄養膜のゲノム増幅の生理的役割と利点は、主にとらえどころのないままです。文化における倍数体栄養膜細胞への二倍体性幹細胞の開発は、このしています<em&gt;生体外で</em&gt;システムの健康と疾患におけるゲノム複製と不安定性の調節機構を解明するための優れたツール。ここでは、チウに発表された変更と、以前の報告に基づいてプロトコルを記述する<em&gt;ら。</em&gt; 2008。

Watch this Scientific Journal Video about Derivation of Mouse Trophoblast Stem Cells from Blastocysts at JoVE.com

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

このビデオでは、我々はマウス胚盤胞と胚盤胞から栄養膜幹細胞の派生の分離を示しています。我々はまた、幹細胞のプロパティのメンテナンスだけでなく、文化の分化誘導のための条件について説明します。

胚盤胞からマウス栄養膜幹細胞の導出

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

derivation-of-mouse-trophoblast-stem-cells-from-blastocysts

Research

JoVE Journal

Biology

18.1K ビュー.  University of Rochester. このビデオでは、我々はマウス胚盤胞と胚盤胞から栄養膜幹細胞の派生の分離を示しています。我々はまた、幹細胞のプロパティのメンテナンスだけでなく、文化の分化誘導のための条件について説明します。

ビデオ: Derivation of Mouse Trophoblast Stem Cells from Blastocysts

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 ...

生物学

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 ...

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 ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

マウスの眼から網膜幹細胞の単離

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

マウスの主栄養膜細胞と絨毛浸潤アッセイの分離

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

長期成体マウスのSSCラインの導出のために文化の精原幹細胞や前駆細胞の濃縮シリアル（SSCS）

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

間葉転換を誘導し、上皮の分析

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

成体マウス切歯から歯科上皮幹細胞の単離および培養

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Methods Article

胚盤胞からマウス栄養膜幹細胞の導出