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.

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</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

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）

生物学

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 ...

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 ...

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

後肢虚血の治療のための胚性幹細胞由来血管内皮細胞

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

ヒト胚性幹細胞のコロニーの成長のビデオバイオインフォマティクス解析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

オリゴデンドロサイト前駆体に胚性幹細胞の分化

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

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 ...

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 ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

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

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

運動ニューロンにマウス胚性幹細胞の効率的な分化

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

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

初代腸オルガノイド培養におけるレトロウイルス感染のビデオプロトコル

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...