胚性幹細胞の分化<ol><li> ESCトリプシン処理によって胚性幹細胞（ESC）の近くにコンフルエントにフラスコを収集する。 </li><li>分化培地を5 mLで細胞を再懸濁し、非ゼラチンコートT25に転送する。 37℃を45分間。上清を取得し、遠心分離を介して細胞を集める。 注：ESC培養物からのマウス胚性繊維芽細胞（MEF）は、非ゼラチンコートT25に容易に接続するため、このめっき工程中に培養液から枯渇している。あなたのMEF上で培養してESCをされていない場合は、事前にプレートをスキップすることができます。 </li><li> 333333 cells/50 mLの分化のメディアで再懸濁し、MEF -枯渇ESC。この濃度は100 ESC/15 mLの結果。 </li><li>使用して、マルチチャンネルピ ​​ペッター、15 cm 2のペトリ皿に100 cells/15 mLのドロップでプレートESC。 8チャンネルピペッターを使用して、プレートあたりの滴の18から22行（プレートあたり5ミリリットル程度）について、合うものでなければなりません。 注：このプロトコルでは、滴の2-5 15センチメートル2皿は十分以上になると2-5 × 10 6 6日目EB由来細胞を得られるはず。 </li><li>ゆっくりと低下を反転させるためにプレートを反転させる。 ° C、5％CO 2で48時間37℃でインキュベートする。 </li><li>プールは、静かにプレートし、15 mLコニカルチューブへの転送を渦巻くことでドロップします。 PBSの4-6 mLでプレートをすすぎ、同じ15 mLコニカルに追加します。 注：最大5つのハンギングドロッププレートまでプールすることがあります。彼らは差別化を続け、彼らはあまりにも集中している場合、彼らは大きな塊を形成する傾向があるとして、EBは成長します。 </li><li>プールされたEBSは重力（約10分）によって沈殿することができます。 EBSを乱すことなく上清を吸引除去する。静かに10mLの分化のメディアと10 cmに転送非接着性のペトリ皿に再懸濁します。 </li><li> 37 50rpmのインキュベートでプレートシェーカー上に置いてペトリ皿° C、5％CO 2。 </li><li> 24時間後（分化4日目）、ペトリ皿の中央にEBSを集中するスワールプレート。慎重に交換5mLの新鮮な分化のメディアとメディアの50％（5mL）を。さらに2日間インキュベーターにプレートを返します。 </li></ol> MSCV - HoxB4 - IRES - GFPとEB解離と感染症<ol><li>分化の6日目に、15 mLコニカルチューブにEBSを転送する。重力によって解決することができます。 </li><li> 10mLのPBSでメディアを再懸濁しますを吸引除去する。 EBSは重力によって再定住することができます。 PBSを吸引除去する。 </li><li> 250mLの解離酵素ミックスし、1 mLのPBSを追加。時折酵素とEBSを混在させるチューブを渦巻いて、20分間37℃の水浴でインキュベートする。 </li><li> 8 mLの酵素を含まない解離緩衝液を追加します。 </li><li> EBSは完全に解離されるまで混合物が（;過度のピペッティングは、EB由来細胞調製物中で死を増加させる約10倍）を5 mLのピペットを使用してひいて粉にする。 <ol><li>混合物は、EBSが解離などの細胞と濁ってくるでしょう。 </li><li>粉砕中にコニカルチューブの底にピペットチップを配置することで大幅に解離を促進するせん断力を作成します。 </li></ol></li><li>遠心分離を介して細胞を集める。 </li><li>再懸濁し、10％IMDM 5mLに細胞をEB由来とトリパンブルー排除を用いてカウント 注：4日目と分化の6日間、EBSは上向きEB由来細胞の30％、6日目で、アポトーシスと細胞のdeath.Thusの大量のどの結果を空洞を作るには、トリパンブルーで染色することがあります。 </li><li>再懸濁し、EB由来の100,000個の細胞における細胞/ 2 mlの10％IMDM +ウイルス上清などそのMOI 5〜10が8 mg / mLの最終濃度に硫酸プロタミンをachieved.Addている。 注：プレート4つの6 -ウェルプレート（ウェル2 mLの/と仮定して）するのに十分なEB /ウイルス上清ミックスを調製する。 </li><li>プレート2 mLのEB /ウイルス上清ミックスウェルあたりのOP9ストローマ細胞（下記参照）で事前にメッキ4個の6ウェルプレート。 </li><li> 90分間室温で2500rpmでプレートを遠心分離します。 37℃インキュベーターにプレートを転送℃、5％CO 2。 </li><li>プレートから上清を4〜6時間後、収穫し、遠心分離を介して懸濁液中に残っている潜在的な細胞を集める。 </li><li> 48 mlの10％IMDM +サイトカインでペレットを再懸濁します（小さくなるかもしれない）。 2 mLの/ウェルの実行された、上清を回収した感染で元の4つの6ウェルプレートに再懸濁を分注する。 注：必ず、使用時に+サイトカイン新鮮な10％IMDMを準備。 </li><li> ° C、5％CO 2七日間37℃培養する。コロニーは4日後の感染と大と7日目では、整形式で明らかなはずである。健全な拡大には/よく7日目に40から60コロニーをもたらす必要があります。 </li><li> 7日目、感染後に、トリプシン処理によりすべてのウェルからのすべてのセルを（OP9間質を含む）の収集とプール。 注：上清またはトリプシンtreatment.Atこの時点文化の中に採用さPBS洗浄を廃棄しないで、いくつかのコロニーは緩く付着している可能性があり、多くの細胞がfloaがありますsuspension.Collectとプールのすべての洗浄上清中のティンは潜在的に価値のあるセルは廃棄されていないことを確認する。 </li><li> 8 mLの新鮮な10％​​IMDMで再懸濁し、細胞+サイトカイン。 four T75フラスコに2 MLS /フラスコを配布します。各フラスコに、13 MLS 10％IMDM +サイトカインを追加。 37 ° C 5％3日間CO 2（10日間の感染後の合計）。 </li></ol> OP9間質の文化とメッキ<ol><li> 20パーセント- MEMでの標準プロトコルにしたがってOP9間質を維持する。コンフルエントに増殖した場合にOP9細胞は、プロパティを変更します。常にそれ以上一時03分より、分割で80％コンフルエントで分割。 </li><li> 24時間前MSCV - HoxB4 - IRES - GFP、20％- MEMで25000 OP9細胞/ウェルのプレート4つの6 - ウェルプレートで6日目EB由来細胞の感染に。 </li></ol>収集、分別及び移植<ol><li>トリプシン処理を経由して十日間OP9ストロマに展開細胞を集めます。細胞を数える。 注意：トリプシン処理の間に用いられる上清またはPBS洗浄を破棄しないでください。文化のこの時点で、いくつかのコロニーは緩く付着している可能性があり、潜在的に価値のあるセルは廃棄されていないことを保証するためにsuspension.Collectとプール内のすべて洗浄し、上清を浮遊し、多くの細胞があります。 注：良い展開が160から200 × 10 6細胞の合計、40〜50 × 10 6細胞/感染オリジナル6穴プレートの間に得 ​​られるはず。 </li><li>細胞は、プロトコルのこの時点では標準的な技術に係る磁気ビーズを選択またはFACSを経由して分画することができる。 </li><li>移植のために、照射2.5時間の9.25 Gyの分割線量を受けるRag-2/gc欠損の受信者（15-22グラムの重さ）に眼窩または尾静脈注射を介して細胞を提供する。離れて。 注：これは動物の体重は、照射の時に15〜22グラムの間に落ちることが重要です。彼らは22グラムより大きい場合、9.25 GYは、適切なアブレーションを確保するために、細胞が致死照射および/ ​​または生着造血コンパートメントから救助を仲介することはできません照射の十分な投与量にはなりません。大型動物を移植する場合、適切な照射線量を実験的に決定する必要があります。 </li><li> 2月5日× 10 6細胞/動物の最低投与量は致死照射から救助を保証するために必要です。細胞を分画する場合、2-5 × 10 6細胞に相当する（ 例えば、関心の人口は、細胞の未分画プールの10％を表す場合は、2-5 × 10 5細胞/動物を注入）注入する。 注：&gt; 2 × 10 6細胞を注入した場合、常に細胞の高い数字が眼窩配信されたときに生じる可能性が、軌道上での奇形腫形成を避けるために、尾静脈を介して注入する。 </li><li>維持RAG - 2 - / - / GC - / -オートクレーブケージの欠乏の受信者。動物施設の&quot;清潔&quot;に応じて、移植後の動物は致死放射線は、次の5週間酸性の水またはTrimethiprim - Sulfasoxazole（Septra）の投与が必要な場合があります。 </li><li> 3週間後の移植では末梢血で&gt; 90％のGFP + ES由来の白血球を期待する。リンパ球がレトロウイルスHoxB4 - IRES - GFPを黙らせるために知られているが生着は、多系統にする必要があります。 </li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>C57Bl/6 Rag-2/gamma-c deficient mice</td>
<td>Animal</td>
<td>Taconic Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plate shaker</td>
<td>Tool</td>
<td>VWR</td>
<td>33998-360</td>
<td>Orbital Shaker by Ikaworks</td>
</tr>
<tr>
<td>Multi-channel pipettor</td>
<td>Tool</td>
<td>VWR</td>
<td>15000-174</td>
<td>ePet by BioHit</td>
</tr>
<tr>
<td>ES trypsin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15090-046</td>
<td>0.25% Trypsin in PBS</td>
</tr>
<tr>
<td>Standard trypsin</td>
<td>Reagent</td>
<td>Invtrogen</td>
<td>25300-062</td>
<td>0.05% Trypsin-EDTA</td>
</tr>
<tr>
<td>PBS</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Enzyme-free dissociation buffer</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>13151-014</td>
<td>&nbsp;</td>
<tr>
<td>Dissociation enzyme mix</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>500 mg Collagenase IV</td>
</tr>
<tr>
<td>Hyaluronidase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H2126</td>
<td>freeze in 500 uL aliquots and avoid repeated freezing and thawing.</td>
</tr>
<tr>
<td>DNAse</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D4527</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10-017CV</td>
<td>&nbsp;</td>
<tr>
<td>Protamine sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P3369</td>
<td>8 ug/mL</td>
</tr>
<tr>
<td>EB differentiation media</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM+cytokines</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>20% anti-MEM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>		</tbody>
		</table>
		</div>

derivation of hematopoietic stem cells from murine embryonic stem cells

幹細胞は、両方の自己複製と生成する複数の分化した子孫の能力を持つ細胞として定義されています。胚性幹細胞（ESC）は、初期胚の胚盤胞に由来し、分化能力に多能性がありますされています。その広大な分化の可能性はそれらの臨床的に有用な細胞型を生成するために、それらを同軸にする方法推理を中心に多くの研究の焦点を行っています。マウスESCからの造血幹細胞（HSC）の成功導出は、最近行われており、このビデオのプロトコルで可視化することができます。 HSC、間違いなく最も臨床的に利用された細胞集団は、造血悪性腫瘍や疾患の無数の治療に使用されています。しかし、HSC治療の恩恵を受ける可能性がある多くの患者は、適切なドナーへのアクセスを欠いている。 ESCは、これらの患者のためのHSCの代替ソースを提供することができます。以下のプロトコルは、ESC - HSCの検討や人間のESCからHSCを分離するための努力を知らせることができるベースラインを確立します。このプロトコルでは、ESCは、市販の血清に対して事前にスクリーニングに最適な造血分化の6日間胚様体（EB）として区別されます。 EBはその後解離し、レトロウイルスHoxB4に感染している。マウス、および10日間のサイトカインを促進する造血の存在下で共培養 - 感染したEB由来細胞はOP9ストローマ、M - CSF - /の頭蓋冠由来する骨髄間質細胞株上にプレーされています。この共培養時には、感染細胞は、世代内のセルの数百万数百の異種プールを結果として、大幅に拡大する。これらの細胞は、致死量放射線照射したマウスを救出して再構成するために使用することができます。

Watch this Scientific Journal Video about Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells at JoVE.com

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

このプロトコルの詳細マウス胚性幹細胞（ESC）と致死量の放射線照射、レシピエントマウスへの彼らのその後の注射から移植造血幹細胞の派生。簡単に言うと、ESCは、レトロウイルスHoxB4とOP9ストローマ細胞と造血サイトカインとの共培養に感染している胚様体、として区別されます。

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-hematopoietic-stem-cells-from-murine-embryonic-stem

Research

JoVE Journal

Biology

13.2K ビュー.  Harvard Medical School. このプロトコルの詳細マウス胚性幹細胞（ESC）と致死量の放射線照射、レシピエントマウスへの彼らのその後の注射から移植造血幹細胞の派生。簡単に言うと、ESCは、レトロウイルスHoxB4とOP9ストローマ細胞と造血サイトカインとの共培養に感染している胚様体、として区別されます。

ビデオ: Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

造血幹細胞の分離と移植（造血幹細胞）

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 Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

プラセンタから造血幹細胞の単離と解析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

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 ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

マウス卵黄嚢とAGMから早期の造血幹細胞の分離

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

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 ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

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 ...

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 ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

マウス造血幹細胞およびリネージュコミット前駆細胞の表現型の解析と分離

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...