This work was supported by a Pilot and Feasibility Grants from the Indiana University School of Medicine Center (IUSM) for Excellence in Molecular Hematology (NIDDK, 5P30DK090948). Additional funding was provided by a Biomedical Enhancement Grant from IUSM. We would like to thank Dr. Karen Cowden Dahl for commenting on the manuscript.

1。胚様体（EB）の形成<ol><li>マウス胚線維芽細胞（MEFを）上で維持されているのESCをゼラチン適応させる。通路のESCのMEFを除去するために0.1％ゼラチンでコーティングした6ウェル組織培養プレートに3回。 <ol><li> （ 表1）未分化の細胞を維持するためにLIFとESCの維持培地中で細胞を成長させる。細胞が80％コンフルエンスを超えないことを確認してください。低継代細胞（MEFから除去した後、10以下の継代）細胞分化のために使用します。 </li></ol></li><li> ESCははEB分化培養する前日に、継代細胞がので、彼らは約50〜70％コンフルエント翌日になります。細胞の多能性を保持するためにESCの維持培地中で細胞を培養し続ける。 </li><li> 6ウェルプレートのウェルあたり：分化、吸引ESCの維持培地との日に1ミリリットルのリン酸緩衝生理食塩水（137mMのNaCl、2.7mMのKCl、11.9 mMリン酸緩衝液、pH7.4 PBS）で洗浄した。 <ol><li> 0.25％tryps200μlのを追加します。EDTA /中各ウェルに加え、3分間37℃でインキュベートする。 </li><li>分化培地を800μlのトリプシン/ EDTAを不活性化する。ブレークアップするための細胞を単一細胞懸濁液にP200チップで2ミリリットル中に上下にピペットで血清学的ピペット。メディアはピペット先端インターフェイス間で上がらないようにP200チップは端にぴったりとフィットしていることを確認してください。 </li><li>上下にピペットで約5〜10倍。ピペッティング時に泡を作成しないように注意してください。 </li></ol></li><li>を15mlコニカルチューブに細胞を移す。 PBSで10ミリリットルにボリュームを起動します。室温で5分間、315×gで遠心分離する。 </li><li>上清を取り除きます。 LIFを含む残りのメディアを削除するには5ミリリットルPBSで2回洗浄する。各洗浄のためのステップ1.4のように遠心分離する。 <ol><li>分化培地（ 表2）2ml中の最終細胞ペレットを再懸濁。 </li></ol></li><li>血球計または細胞カウンターで細胞を計数10cmのペトリ皿中6,000-10,000細胞/ mlをプレート（非組織培養処理）。 <ol><li>各ESC株について経験的に正確な細胞/ mlを決定します。通常、細菌仕事や低付着プレートに使用する無菌のペトリ皿を使用してください。 注：最良の分化の結果を得るには、EBをプレートに付着することなく、懸濁液中に残っている球体を形成すべきである。少なくともアドヒアランスを持つものを見つけるために、プレートのいくつかのブランドをテストします。 </li><li> 5％CO 2、37℃の組織培養インキュベーター中で分化するのESCをインキュベートする。 </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0"><colgroup><col /><col span="2" /><col /><col /><col /></colgroup><tbody><tr><td> 試薬 </td><td> ストック </td><td> 最終濃度 </td><td> ボリューム </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>のDMEM </td><td> 1倍</td><td></td><td> 410ミリリットル</td><td> S<html> igma /アルドリッチ</td><td> D5796 </td></tr><tr><td> FBS（ES選抜し） </td><td> 100％ </td><td> 15％ </td><td> 75ミリリットル</td><td>ハイクローン</td><td> SH30070.03E </td></tr><tr><td>非必須アミノ酸</td><td> 100X </td><td> 1倍</td><td> 5ミリリットル</td><td>ライフテクノロジーズ</td><td> 11140 </td></tr><tr><td> L-グルタミン</td><td> 100X </td><td> 1倍</td><td> 5ミリリットル</td><td>ライフテクノロジーズ</td><td> 35050 </td></tr><tr><td> Penncillin /ストレプトマイシン</td><td> 100X </td><td> 1倍</td><td> 5ミリリットル</td><td>ライフテクノロジーズ</td><td> 15070 </td></tr><tr><td> βメルカプトエタノール</td><td> 14.3 M </td><td> 114μM </td><td> 4μlの</td><td>シグマ/アルドリッチ</td><td> M3148 </td></tr><tr><td>白血病抑制因子（LIF） </td><td> 10 7単位/ ml </td><td>千単位/ ml </td><td> 50μlの</td><td>ミリポア</td><td> ESG1107 </td></tr></tbody></table>ontent &quot;&gt; 表1：ESC保守メディア。 <table border="0" cellpadding="0" cellspacing="0"><colgroup><col /><col span="2" /><col /><col span="2" /></colgroup><tbody><tr><td> 試薬 </td><td> ストック </td><td> 最終濃度 </td><td> ボリューム </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>のDMEM </td><td> 1倍</td><td></td><td> 410ミリリットル</td><td>シグマ/アルドリッチ</td><td> D5796 </td></tr><tr><td> FBS（ユーザーは、最適な分化についてスクリーニング） </td><td> 100％ </td><td> 15％ </td><td> 75ミリリットル</td><td>ハイクローン</td><td> SH30070.03E </td></tr><tr><td>非必須アミノ酸</td><td> 100X </td><td> 1倍</td><td> 5ミリリットル</td><td>ライフテクノロジーズ</td><td> 11140 </td></tr><tr><td> L-グルタミン</td><td> 100X </td><td> 1倍</td><td> 5ミリリットル</td><td>ライフ【分析シナジー</td><td> 35050 </td></tr><tr><td> Penncillin /ストレプトマイシン</td><td> 100X </td><td> 1倍</td><td> 5ミリリットル</td><td>ライフテクノロジーズ</td><td> 15070 </td></tr><tr><td> βメルカプトエタノール</td><td> 14.3 M </td><td> 114μM </td><td> 4μlの</td><td>シグマ/アルドリッチ</td><td> M3148 </td></tr></tbody></table> 表2：分化培地。 ウイルス感染のための2の準備EB細胞<ol><li> （2日目と3日目の間）、開発の目的の段階では、15ミリリットルコニカルチューブに10センチメートルペトリ皿からのEBを転送します。 5ミリリットルのPBSでプレートを洗浄し、円錐管に洗浄を追加します。 </li><li> 5分間室温で315×gでのEBをペレット。 5ミリリットルのPBSでEB細胞を洗浄。 </li><li> （タンパク質分解およびコラーゲン分解酵素を含む）解凍した細胞剥離溶液1mlを加え、37℃の水浴（またはインキュベーター）内チューブを配置OCCで30分間（フリックまたは軽ボルテックスから）asionalに攪拌。 </li><li>分化培地の1ミリリットルを追加します。ピペットで上下にP200ピペットチップで2ミリリットルをピペットで。 30〜60分間、再び37℃の水浴中にチューブを置きます。 </li><li> 5分間315×gで細胞をスピンダウンし、PBSで細胞を洗浄。 </li></ol> 3。ウイルス感染<ol><li>分化培地1.5 mlの細胞を再懸濁。 6ウェル非組織培養処理プレートに細胞懸濁液を移す。 </li><li>事前に標準的な技術によって、レトロウイルスを準備します。ウイルス力価に応じて、細胞へのウイルス上清の5から100を添加する。これはEBのその後の改革を阻害するとして、感染を強化するためにポリブレンを追加しないでください。 </li><li>室温で90分間、2,120×gで遠心分離することによってウイルスで細胞をSpinoculate。 </li></ol> 4ハンギングドロップでウイルス感染のEB細胞を分化<ol><li>私の各ウェルに分化培地2 mlを加えnfected EB細胞懸濁液。 </li><li>ピペットでmultipipettorsための滅菌試薬槽への感染のEB細胞懸濁液3.5ミリリットル。 820μlの15〜20行をピペットするためにマルチピペッターを使用して、15センチメートルペトリ皿の反転蓋の上にドロップします。 </li><li>ペトリ皿の下半分に、滅菌PBSまたは水10mlを追加します。その後、ペトリ皿の上に落下し、市町村と蓋を反転。滴が、チャンバの加湿を保つために逆さまにPBSで蓋または下記の水からぶら下がっていることを確認します。 </li><li> 5.0％CO 2、37℃の組織培養インキュベーターに皿を置き。 </li><li> 2日後、吊り内に形成されたEBは、分化培地中の4.5 mlの蓋と洗浄を反転させることにより低下集める。 <ol><li>新しい10センチメートル非組織培養ペトリ皿へ伝達媒体とのEB。分化培地と10cmプレートへの転送3ミリリットルでもう一度蓋を洗ってください。 </li></ol></li><li> 8の合計の後にフローサイトメトリーによる分析のために細胞を回収する日間の培養は、分化培地（-LIF）への細胞の初期転送から開始する。 </li></ol>フローサイトメーター分析のための5。の準備細胞<ol><li> 10ミリリットルピペットで15ミリリットルコニカルチューブに細胞を移す。 5ミリリットルのPBSでプレートを洗浄し、同じ円錐チューブに移す。 </li><li> 315×gで細胞をペレット化。 10ミリリットルのPBSでペレットを洗浄する。 </li><li>解凍した細胞剥離溶液1mlを加え、時折撹拌しながら30分間37℃の水浴中にチューブを置き。いくつかの細胞表面抗原の検出のために、トリプシンを用いてもよい。 </li><li> 1ミリリットル分化培地を追加し、ピペッティングし、2mlピペットでダウン。助けるためにピペットの先端にP200チップを使用ブレークアップ単一細胞懸濁液にEBを消化した。 <ol><li>内在性膜タンパク質は、細胞表面にリサイクルするために60分間再び37℃の水浴中に管を置き。 </li></ol></li><li> 5分間315×gで細胞をペレット化。 </li><li>細胞w iを洗う第0.1％ウシ血清アルブミンフラクションV（BSA）をPBS（PBS / BSA）中で調製し、特異的な蛍光タグ化抗体で細胞をインキュベートする。 </li></ol>造血前駆細胞のための6。フローサイトメトリー分析<ol><li> 5ミリリットル12×75ミリメートルの丸底チューブ中の10 6のEB由来細胞を配置します。補償の設定フローサイトメーターを調整するために、製造者の指示に従って補償ビーズを使用しています。 </li><li> 315×gで細胞をペレット化。チューブ内の残留PBS / BSA溶液中で上清と再懸濁ペレットをオフに注ぐ。 </li><li> ESCのHPCを検出するために、蛍光標識抗体で細胞をインキュベートする：抗マウスCD41-PE（R-フィコエリトリン）および抗マウスCD117（cKitの）-APC / CY7（アロフィコシアニン7）。 <ol><li> PBS / BSA中100：抗体1に希釈する。各サンプルに希釈した抗体を100μlを加える。各ベンダー、クローン、ロット又は抗体に対する抗体の正確な希釈を決定します。個別の細胞を標識するための最適な希釈を決定します。 </李&gt; </ol></li><li>暗所で30分間氷上でサンプルをインキュベートする。 </li><li> 5分間315×gで2ミリリットルのPBS / BSAおよびペレットの細胞を追加します。 </li><li> 300のPBSμlあるいは他の等張緩衝液中に吸引し上清とペレットを再懸濁し。 </li><li>大きな細胞凝集体を除去するためにセルストレーナーキャップ（0.35μM）を介して再懸濁した細胞をフィルタリングします。 </li><li>フローサイトメーターで細胞を分析します。 </li></ol>

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M., Passegue, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+controlling+hematopoietic+stem+cell+functions+during+normal+hematopoiesis+and+hematological+malignancies.+Wiley+interdisciplinary+reviews.">Mechanisms controlling hematopoietic stem cell functions during normal hematopoiesis and hematological malignancies. Wiley interdisciplinary reviews.</a> Systems biology and medicine. 3, 681-701 (2011).</li><li>Dahl, R., Ramirez-Bergeron, D. L., Rao, S., Simon, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spi-B+can+functionally+replace+PU.1+in+myeloid+but+not+lymphoid+development.">Spi-B can functionally replace PU.1 in myeloid but not lymphoid development.</a> Embo J. 21, 2220-2230 (2002).</li><li>Dahl, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+macrophage+and+neutrophil+cell+fates+by+the+PU.1:C/EBPalpha+ratio+and+granulocyte+colony-stimulating+factor.">Regulation of macrophage and neutrophil cell fates by the PU.1:C/EBPalpha ratio and granulocyte colony-stimulating factor.</a> Nat Immunol. 4, 1029-1036 (2003).</li></ol>

著者らは、開示することなく、競合する金融利害関係はありません。

上述したように、誘導的に目的とする遺伝子を発現ESCクローンはドキシサイクリンシステムを使用して生成することができるが、これらの株の生成は、時間がかかり、労働集約的である。このプロトコールに記載されたEB由来ESCから調製単一細胞懸濁液中の目的の遺伝子を発現する方法である。これらの感染した細胞は、その後の分化を調べるために、ドロップを掛けてEBをに改質されている...

Murine embryonic stem cells (ESCs) are pluripotent, remaining undifferentiated and self-renewing in the presence of the cytokine Leukemia Inhibitory Factor (LIF)1. Upon withdrawal of LIF they will spontaneously differentiate into 3-dimensional (3D) structures called embryoid bodies (EBs)2. The 3D architecture allows for the development of the three germ layers ectoderm, endoderm, and mesoderm, which then later give rise to mature tissue types3. ESCs are an exceptional model for elucidating the molecular mechanisms of cellular differentiation, particularly the investigation of the development of early hematopoietic progenitor cells (HPCs)4. 
Gene expression in ESCs can be manipulated by several techniques that allow for the determination of a role for individual molecules in development. One of the most common techniques is to use homologous recombination to generate ESC lines, which lack a gene of interest5,6. There are also a number of techniques that have been used to overexpress genes. The first technique used to modify gene expression in ESCs was to infect them with recombinant retroviruses7,8. The gene of interest however is often silenced as the ESCs differentiate into progenitor and mature cell types. Use of lentiviruses has been successful in limiting the silencing of virally expressed genes9. Other viral vectors used for overexpression include adenovirus and adeno-associated virus10. In addition standard transfection techniques to stably introduce expression plasmids are widely used for ESC transgene expression11. One difficulty with these systems is that often expression of a specific gene has different effects depending on its temporal expression. For example, the Smad1 protein affects the development of hematopoietic cells differently during different stages of EB development12,13. 
This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest. The most common system for inducibly expressing transgenes in ESCs uses the tetracycline resistance operon from E. Coli (Escherichia coli). Several different tetracycline systems have been developed. One of the more popular strategies was developed by Kyba and colleagues14. They generated an ESC line (Ainv15), which has the reverse tetracycline transactivator gene inserted into the constitutively active ROSA26 locus. A tetracycline response element (TRE), a downstream LoxP site (locus of X-over P1 from bacteriophage P1), and a promoterless neomycin cassette were introduced into the HPRT (Hypoxanthine-guanine phosphoribosyltransferase) locus on the X chromosome. Using a CRE recombination approach a gene of interest along with a eukaryotic promoter to drive the expression of the neomycin resistance gene can be inserted into the LoxP site. Correctly targeted ESCs are isolated by G418 selection. These targeted clones then must be tested for doxycycline (or tetracycline) inducible expression of the transgene. This approach has successfully generated ESC lines that inducibly express HoxB4, Stat5, SCL, and Smad714-17. However, generation of these inducible cell lines is time consuming. Described here is a method to disaggregate ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infect the cell suspensions at different days of development, and then reform the EBs by hanging drop. Downstream differentiation is later evaluated by flow cytometry. In this article an example of how miRNA expression in EB-derived cells effects hematopoietic differentiation is shown. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

<table><tbody><tr><td>DMEM</td><td>Sigma/Aldrich</td><td>D5796</td><td></td></tr><tr><td>&amp;#946;-mercaptoethanol</td><td>Sigma/Aldrich</td><td>M3148</td><td></td></tr><tr><td>FBS (ES Screened)</td><td>Hyclone</td><td>SH30070.03E</td><td></td></tr><tr><td>FBS (Defined)</td><td>Hyclone</td><td>SH30070.03</td><td></td></tr><tr><td>Non-essential Amino Acids</td><td>Life Technologies</td><td>11140</td><td></td></tr><tr><td>L-Glutamine</td><td>Life Technologies</td><td>35050</td><td></td></tr><tr><td>Penn/Strep</td><td>Life Technologies</td><td>15070</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life Technologies</td><td>25200</td><td></td></tr><tr><td>LIF ESGRO</td><td>Millipore</td><td>ESG1107</td><td></td></tr><tr><td>ACCUMAX</td><td>Millipore</td><td>SCR006</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Life Technologies</td><td>25200</td><td></td></tr><tr><td>Falcon Tissue Culture Plates 6-well</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>Falcon Non-treated Plates 6-well</td><td>Fisher Scientific</td><td>08-772-49</td><td></td></tr><tr><td>Falcon Petri dish 10 cm</td><td>Fisher Scientific</td><td>08-757-100D</td><td></td></tr><tr><td>Falcon Petri dish 15 cm</td><td>Fisher Scientific</td><td>08-757-148</td><td></td></tr><tr><td>Falcon Tube 5 ml</td><td>Fisher Scientific</td><td>14-959-11A</td><td></td></tr><tr><td>Falcon Tube 5 ml with Cell Strainer Cap</td><td>Fisher Scientific</td><td>08-771-23</td><td></td></tr><tr><td>White sterile resevoirs</td><td>U.S.A. Scientific</td><td>111-0700</td><td></td></tr><tr><td>Rat anti-mouse CD41 PE-conjugated</td><td>Biolegend</td><td>133906</td><td></td></tr><tr><td>Rat anti-mouse CD117 APC/CY7-conjugated</td><td>Biolegend</td><td>105826</td><td></td></tr><tr><td>RW4 ESCs</td><td>ATCC</td><td>CRL-12418</td><td></td></tr></tbody></table>

retroviral infection of murine embryonic stem cell derived embryoid body cells for analysis of hematopoietic differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

これらの研究ではESCを使用した（129X1 / SVJマウス系統由来）RW4を糊化。 EBを分化の3日後に単離した。最良の結果を得るにはEBを球状と非接着性（ 図1A、B）である必要があります。ウイルスは、目的の遺伝子と一緒に蛍光マーカーGFPを共発現したピノキュレーションした後、EBをハンギングドロップ法により改質された。 EBを成功裏に2.0、2.5、および3.0日EBから調製した細胞懸濁液?...

Watch this Scientific Journal Video about Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation at JoVE.com

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

マウス胚のレトロウイルス感染は、造血分化の解析のための細胞由来の胚様体幹細胞

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

retroviral-infection-murine-embryonic-stem-cell-derived-embryoid-body

Research

JoVE Journal

Biology

8.4K ビュー.  Harper Cancer Research Institute. Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation. 

ビデオ: Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

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

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

マウス胚性造血内皮細胞の単離

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

発生生物学

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

転写因子との再プログラミングマウス胚線維芽細胞造血プログラムを誘導するために

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

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 ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

骨髄前駆細胞のレトロウイルス形質導入は、T細胞受容体Retrogenicマウスを生成するために、

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

免疫・感染学

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

レンチウイルスを介したノックダウン中生体外で</emヒト造血幹細胞の>赤血球

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (c&#947;-/-)-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility.

This protocol describes the methods in constructing a humanized bone-marrow/liver/thymus mouse model with stem cell-based engineered immunity against HIV infection.

ヒト化マウスモデルにおけるHIV感染に対する幹細胞ベースの設計さイミュニティ

With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic ...

医学

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 ...

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

疾患モデルにおける血化細胞の研究のためのレンチウイルスCRISPR/Cas9媒介ゲノム編集

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

幹細胞由来ウイルスAg特異的Tリンパ球はマウスにおけるHBV複製を抑制する

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...