我々は、貴重なアドバイスを信行紀夫、仁滝沢とマルクスマンツに感謝します。この作品は、スイス国立科学財団、スイスのがんリーグとFondazione Ticinese当たりラ未公表スルCancroによって資金を供給された。

1。骨髄からの細胞懸濁液の調製<ol><li>マウスを安楽死させると、その腹部と背面にステンレスパンとスプレー70％エタノール中に動物を配置します。後ろ足、と脊柱からの大腿骨と脛骨を収集します。 </li><li>正確に鋭い先端をハサミでとガーゼで足の骨から脊柱からすべての軟組織の残留物を除去する。 50μMβ-メルカプトエタノールを添加した10％の熱不活性化ウシ胎児血清（FBS）を含むRPMI培地30mLを、100μMのMEM非必須アミノ酸、1 mMのMEMピルビン酸ナトリウム、2 50mLのファルコンチューブに店の骨mM L-グルタミン、100μg/ mlのストレプトマイシン、100 U / mlペニシリン。 </li><li>滅菌フィルターで予め滅菌乳鉢の底をカバーしています。 </li><li>モルタルに骨を移し、摩擦面を組み立てるために、フィルターの層でそれらをカバーしています。 </li><li>ファルコンフィルタが設けられた50mLのファルコンチューブを準備します。 </li><li>骨を砕く均一な細胞懸濁液が得られるまで乳棒の助けを借りて。 </li><li>チューブに懸濁液を移す。 </li><li>残りの細胞を洗浄し、収穫する新鮮なRPMI完全培地中のモルタル5mLに追加します。 </li><li>均質化された骨が明らかに表示されるまで、最後の2点を繰り返します。 </li><li>残りの組織片を除去するために細胞懸濁液を移し、新しいチューブにフィルタリングします。 </li><li> 5分間1,500 rpmでチューブを遠心分離し、慎重に吸引して上清を除去します。細胞ペレットを乱さないようにしてください。 </li></ol> 2。表現型分析<ol><li> PBS、2％FBS 15mLに個々のマウスの細胞を再懸濁し、洗浄してください。 </li><li> 5分間1,500 rpmでチューブを遠心分離し、慎重に吸引して上清を除去します。ペレットを乱さないことを確認してください。 </li><li> 5 mLのPBS、2％FBSおよびcount細胞のペレットを一時停止します。 </li><li>転送1×10 6細胞/ウェル、96ウェル丸底でプレート。 </li><li> 5分間1,500 rpmでプレートを遠心分離します。 </li><li>以下の表面マーカーに対するモノクローナル抗体のミックスを調製する。それらの細胞を染色するためにPBS、2％FBSに示されている希釈します。 <ul type="disc"><li> PE-Cy5で1:200に結合したCD4、CD8、CD3、CD45R、CD19、GR1、TER119、NK1.1 </li><li> APC 1:100に結合したc-Kitの</li><li>ビオチン1:50〜共役SCA1 </li><li> FITC 1:50〜共役CD34 </li><li> PE 1:100に結合したFcγRと</li><li> PE-Cy7 1:100に結合したIL-7R </li></ul></li><li>上清を捨て、各ウェルにモノクローナル抗体ミックス50μLを追加します。ピペッティングにより単一細胞に解離する。 </li><li> 4℃で暗所で20分間細胞をインキュベート</li><li> PBS150μLの2％FBSで細胞を2回洗浄します。 </li><li> 5分間1500rpmでプレートを遠心分離し、反転板により上清を捨てる。 </li><li> PBS中でストレプトアビジンを希釈し、2％FBSは、細胞を染色することが示された。 <ul type="disc"><li>ストレプトアビジンコンジュAPC-Cy7 1:300にゲート</li></ul></li><li>各ウェルに溶液50​​μLを追加します。ピペッティングによって単一細胞懸濁液に解離する。 </li><li> 4℃で暗所で10分間細胞をインキュベート</li><li> PBS150μLの2％FBSで細胞を2回洗浄します。 </li><li> 5分間1,500 rpmでプレートを遠心し、上清を捨てる。 </li><li> PBS、2％FBSを100μLに細胞を再懸濁します。 </li><li> FACSでサンプルを取得します。 </li></ol> 3。のc-Kit +細胞の磁気濃縮<ol><li> PBS、pHは7.2、（BSA）0.5％ウシ血清アルブミン、および2mM EDTAを含む緩衝液を準備します。 </li><li>骨の細胞ペレットを再懸濁し1:50に希釈し、APCに結合したc-KitのmAbを含む緩衝液1mLに少なくとも10匹のマウスから派生した髄。 </li><li> 4℃で暗所で20分間細胞をインキュベート</li><li>緩衝液40mLで2回細胞を洗浄します。 </li><li> 5分間1,500 rpmでチューブを遠心分離し、慎重にtを削除する彼は、吸引によって上清。ペレットを乱さないことを確認してください。 </li><li>再懸濁し、細胞ペレットとペレットに直接各安楽死させたマウスの抗APC MicroBeadsは50μlを加える。 </li><li>よく混ぜ、4℃で暗所で20分間インキュベート</li><li> 5分間1,500 rpmで緩衝液、遠心分離を40 mLを加えることにより細胞を洗浄します。完全に上清を吸引除去する。 </li><li>緩衝液4 mLの安楽死させたすべての3匹のマウスの複数の再懸濁し、細胞ペレット。 </li><li>適当なのMACS Separatorの磁場内の場所MACS LSカラム。すべての3匹安楽死させた1列を使用しています。 </li><li>緩衝液4 mLで洗浄してカラムを準備します。 </li><li>カラムに細胞懸濁液を適用します。 </li><li>を通過し、緩衝液4 mLでカラムを洗浄し非標識の細胞を収集します。緩衝液4 mLの三回を追加することによって、洗濯の手順を実行します。カラムのリザーバーが空にされた新しいバッファを追加します。 </li><li>セパレータとPLACから列を削除します。電子15mlファルコンチューブの上に各1。 </li><li>各カラムに緩衝液をピペットで4mlの。すぐに列にプランジャーを押すことにより、磁気標識された細胞を洗い流す。 </li><li> 5分間1,500 rpmでチューブを遠心し、注意深く吸引により上清を除去します。 </li><li>シングル15mlファルコンチューブに緩衝液およびグループのペレットを1 mLの各ペレットを再懸濁します。 </li></ol> 4。造血幹細胞およびリネージュコミットされた前駆細胞の選別<ol><li>以下の表面マーカーに対するモノクローナル抗体のミックスを調製する。それらの細胞を染色するために500μLのPBS、2％FBSに示されている希釈します。 <ul type="disc"><li> PE-Cy5で1:200に結合したCD4、CD8、CD3、CD45R、CD19、GR1、TER119、NK1.1 </li><li> APC 1:100に結合したc-Kitの</li><li>ビオチン1:50〜Sca-1は共役</li><li> FITC 1:50〜共役CD34 </li><li> PE 1:100に結合したFcγRと</li></ul></li><li>先行するからチューブを遠心sは5分間1500 rpmでセクションを注意深く吸引して上清を除去します。 </li><li>ペレットにモノクローナル抗体ミックスを追加します。ピペッティングによって単一細胞懸濁液に解離する。 </li><li> 4℃で暗所で20分間細胞をインキュベート</li><li> PBS、2％FBSの14mLで2回細胞を洗浄します。 </li><li> 5分間1,500 rpmでチューブを遠心し、上清を捨てる。 </li><li>ストレプトアビジンなどの細胞を染色するために500μLのPBS、2％FBSに示されている希釈します。 <ul type="disc"><li> APC-Cy7 1:300に結合したストレプトアビジン</li></ul></li><li>チューブへの解決策を追加します。ピペッティングによって単一細胞懸濁液に解離する。 </li><li> 4℃で暗所で10分間細胞をインキュベート</li><li> PBS 14 mLおよび2％FBSで2回細胞を洗浄します。 </li><li> 5分間1,500 rpmでチューブを遠心し、上清を捨てる。 </li><li> PBS、2％FBSを500μLに細胞を再懸濁します。 </li><li>イーライするために、新しい5 mlチューブに細胞懸濁液を移し、フィルタリングソート手順を妨げる可能性の細胞凝集体をminate。 </li><li>ソートされた集団から7-AAD +死細胞を除外するために、細胞懸濁液に7-AADの5μL（それは1:100に希釈し使用）を追加します。 </li><li>以下の表現型に基づいてBD FACSAriaでソート細胞： <ul type="disc"><li> LKS細胞：LIN -のc-Kit + Sca-1は+ </li><li> LT-造血幹細胞：LKS CD34 - </li><li> ST-造血幹細胞：CD34 + LKS </li><li> GMPに：LIN -のc-Kit + Sca-1は低 FcγRと高い CD34 + </li><li>欧州議会：LIN -のc-Kit + Sca-1は低 FcγRと低い CD34 - </li><li>のCMP：LIN - CD34のc-Kit + Sca-1は低 FcγRと低い+ </li></ul></li></ol> 5。代表的な結果 1に示すように、電子ゲー手順の例を図スコアリンなどの一般的なリンパ球前駆細胞（CLPの） - / c-KitのLO /インターロイキン（IL）-7R +細胞;林- / c-Kitの+ / SCA-1 +（LKS）とリン- / c-Kitの+ / SCA -1 LO（のc-Kit +）細胞は、c-Kitの+ゲートから、共通の骨髄前駆細胞（CMPの）はFcγRとLO CD34 +細胞は、FcγRとハイ CD34 +細胞および/ ​​巨核球などの顆粒球/単球前駆細胞（GMPに）として識別されます。 FcγRとLO CD34などの赤血球前駆細胞（MEPS） -細胞; -それぞれとCD34 +細胞、LKSゲート、LT-HSCとST-HSCからCD34として同定されています。その後、上記の表現型に基づいてCD34の発現だけでなく、他の前駆細胞に応じて並べ替えることができLT-HSCとST-HSC、この骨髄細胞懸濁液を磁気ビーズ（ 図2）のc-Kit +細胞を濃縮することができます。 HSCはANAになります<html> 図4に示すように、ライブイメージング共焦点顕微鏡で溶菌ここで、CD34 -細胞はヌクレオチド結合化合物キナクリン11と同様に核赤（DRAQ5）で染色した。造血幹細胞ではなく、GMPには、キナクリン陽性小胞は細胞質12に表示されます。 + LT-HSCの暴露プリンP2受容体の活性化による細胞内Ca 2の増加を示し、図5に示すようにまた、ソートされた造血幹細胞は、機能の実験で使用することができアデノシン三リン酸（ATP）とイオノマイシン12。 <img alt="図1" src="/files/ftp_upload/3736/3736fig1.jpg" /> 図1 LIN-BM細胞の代表的なドットプロット解析。プロトコルのテキストで説明したように骨髄細胞懸濁液から造血前駆細胞と造血幹細胞を識別し、得点するためのフローサイトメーターでの電子ゲートの手順では、染色した。 pload/3736/3736fig2.jpg &quot;/&gt; 図2骨髄からのc-Kit +細胞の選択的濃縮のためのスキーム。 <img alt="図3" src="/files/ftp_upload/3736/3736fig3.jpg" /> 図3電子ゲートLKS CD34の細胞周期分析-健常者とKi-67抗体およびDAPIで染色したIBDを有するマウスにおける造血幹細胞。細胞が溶解/修正し、1μg/ mlでFITCコンジュゲートのKi-67およびDAPIで染色し、続いてバッファーをパーマで固定し、浸透させた。 LKS CD34内のすべての検出可能であればサイクリング細胞がほとんどです-健常者の造血幹細胞コンパートメント12。 <img alt="図4" src="/files/ftp_upload/3736/3736fig4.jpg" /> 図4 CD34をソートFACSのイメージング共焦点顕微鏡をライブ- HSCとGMPには、ヌクレオチド結合化合物のキナクリンと核赤（DRAQ5）で染色した。小さいの象限は、キナクリンposを表示itive小胞は造血幹細胞の細胞質内に検出されましたが、GMPにはありません。 <img alt="図5" src="/files/ftp_upload/3736/3736fig5.jpg" /> 図5。ソートされた造血幹細胞における細胞内Ca 2 +上昇がフラ-2をロードおよびATPとイオノマイシンで刺激した。単一細胞からのトレースが表示されます。すべての細胞は細胞外ATPは細胞質ゾルのCa 2の顕著な増加を示す+ ATPの添加後に反応した。

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利害の衝突が宣言されません。

ここで説明する方法は、個々のマウス（ 図1）における造血の迅速かつ正確な分析を可能にします。マウス炎症モデルでは、自己免疫疾患、免疫不全、変性疾患、代謝性疾患および癌を含む様々な実験設定ではこの分析は、造血における病理学的条件の影響に対処することができます。 図3は、電子的にゲートLKSにおける細胞周期活性の分析を示しています。 </su...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> RPMI 1640 </td><td>ギブコ</td><td> 42401 </td></tr><tr><td> MEM NEAA 100X </td><td>ギブコ</td><td> 11140 </td></tr><tr><td>ピルビン酸ナトリウム</td><td>ギブコ</td><td> 11360 </td></tr><tr><td> PenStrep </td><td>ギブコ</td><td> 15070 </td></tr><tr><td> PBS </td><td>ギブコ</td><td> 20012 </td></tr><tr><td> FBS </td><td>ギブコ</td><td> 16000 </td></tr><tr><td>セルストレーナーは40μm </td><td> BDファルコン</td><td> 352340 </td></tr><tr><td> 7-AAD染色液</td><td> BD Pharmingen社</td><td> 559925 </td></tr><tr><td>バッファを溶解/修正</td><td> BD Pharmingen社</td><td> 558049 </td></tr><tr><td>バッファIIIをパーマ</td><td> BD Pharmingen社</td><td> 558050 </td> &lt;/ TR&gt; <tr><td> Ki-67の</td><td> BD Pharmingen社</td><td> 556026 </td></tr><tr><td> DAPI </td><td>インビトロジェン</td><td> D21490 </td></tr><tr><td> CD4（GK1.5） </td><td>ベイバイオサイエンス</td><td> 150041 </td></tr><tr><td> CD8（53から6.7） </td><td>ベイバイオサイエンス</td><td> 150081 </td></tr><tr><td> CD3（145-2C11） </td><td>ベイバイオサイエンス</td><td> 150031 </td></tr><tr><td> CD45R（RA3-6B2） </td><td>ベイバイオサイエンス</td><td> 150452 </td></tr><tr><td> CD19（6D5） </td><td>ベイバイオサイエンス</td><td> 150193 </td></tr><tr><td> GR1（RB6-8C5） </td><td>ベイバイオサイエンス</td><td> 155931 </td></tr><tr><td> Tre119（TER-119） </td><td>ベイバイオサイエンス</td><td> 155921 </td></tr><tr><td> NK-1.1（PK136） </td><td>ベイバイオサイエンス</td><td> 455941 </td></tr><tr><td>のc-Kit（2B8） </td><td>ベイバイオサイエンス</td><td> 171171 </td></tr><tr><td> SCA-1（D7） </td><td> eBiosciENCE </td><td> 135981 </td></tr><tr><td> CD34（RAM34） </td><td>ベイバイオサイエンス</td><td> 110341 </td></tr><tr><td> FcγRと（2.4G2） </td><td>ベイバイオサイエンス</td><td> 553145 </td></tr><tr><td>抗APC MicroBeadsは</td><td>ミルテニーバイオテク</td><td> 130-090-855 </td></tr><tr><td> LSカラム</td><td>ミルテニーバイオテク</td><td> 130-042-401 </td></tr></tbody></table>

phenotypic analysis and isolation of murine hematopoietic stem cells and lineage-committed progenitors

骨髄は、造血幹細胞とより成熟した血液細胞系列の前駆細胞が存在すると成人生体内で分化する主要なサイトです。造血幹細胞は、生命時間1のすべての血液細胞系列を生成することができる多能性細胞の分細胞集団を構成しています。骨髄中の造血幹細胞恒常性の分子解剖は、造血、腫瘍学、再生医療において重要な意味を持っています。我々は個々のマウス（ 図1）造血前駆細胞サブセットと造血幹細胞の分布を獲得するためにフローサイトメトリーで蛍光抗体、電子ゲートの手順でラベルプロトコルを記述します。加えて、我々は広範囲に造血前駆細胞と同様に長期的（LT）およびc-Kitを発現する細胞の磁気濃縮によりプールされた骨髄細胞懸濁液から造血幹細胞を再構成する短期（ST）を豊かにする方法について説明します。得られた細胞調製物は、in vitroで選択したサブセットをソートするために使用することができ、生体機能研究である（ 図2）。 両方の海綿骨芽細胞2,3と類 ​​洞内皮4は、骨髄中の造血幹細胞をサポートする機能的なニッチを構成しています。そのリガンドであるアンジオポエチン-1 5とN-カドヘリン+骨芽細胞3とHSCで発現する受容体チロシンキナーゼTie2の相互作用のサブセットを含む、骨芽細胞ニッチのいくつかのメカニズムは、造血幹細胞の休止を決定する際に同意する。骨髄中の&quot;休止状態&quot;は、レプリケーションや過度のサイクリング活動の6時の最終的な疲労から造血幹細胞を保護するために重要である。そのようなToll様受容体リガンドとしての自然免疫系の細胞7、インターフェロン-α6に作用する外因性の刺激はまた系統コミットされた前駆細胞に造血幹細胞の増殖と分化を誘導することができる。最近では、LIN内で休眠中のマウスの造血幹細胞の集団-のc-Kit + Sca-1は+ CD150 + CD48 - CD34 -人口が8に記載されています。ここで説明したように造血前駆細胞に富む細胞懸濁液からCD34の発現に基づいてセルの並べ替えは、静止自己再生LT-HSCとST-HSCの9の両方の分離を可能にします。系統陽性細胞の枯渇とCD48とFlk2抗体とLT-HSCのソートに基づいて同様の手順では、以前に10に記載されている。本報告では、表現型の特性と異なる生理学的および病理学的条件下で造血を監視するために有用である造血前駆細胞のex vivoでの細胞周期解析のためのプロトコルを提供しています。さらに、細胞生物学、シグナル伝達アッセイと同様に移植のために彼らの自己再生、拡大と分化を制御する要因とメカニズムを定義するために使用することができ造血幹細胞、の手順をソートFACSを説明します。

Watch this Scientific Journal Video about Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors at JoVE.com

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

フローサイトメトリーにおける骨髄造血前駆細胞の分布と同様に効率的に分離するために高度に精製された造血幹細胞（HSC）を分析する方法が記載されている。アイソレーション·プロシージャは、本質的には、c-Kit +細胞と細胞および分子研究のための造血幹細胞を精製するために細胞選別の磁気濃縮に基づいています。

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 ...

phenotypic-analysis-isolation-murine-hematopoietic-stem-cells-lineage

Research

JoVE Journal

Biology

18.5K ビュー.  Bellinzona (Switzerland). フローサイトメトリーにおける骨髄造血前駆細胞の分布と同様に効率的に分離するために高度に精製された造血幹細胞（HSC）を分析する方法が記載されている。アイソレーション·プロシージャは、本質的には、c-Kit +細胞と細胞および分子研究のための造血幹細胞を精製するために細胞選別の磁気濃縮に基づいています。

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

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 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. ...

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 ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

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

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

間葉転換を誘導し、上皮の分析

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

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

筋衛星細胞の単離、培養、および移植

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

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.

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 ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

RNAシーケンシング解析によりEML造血前駆細胞における自己再生および分化を調節する重要な要因の同定

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

造血幹細胞とT細胞におけるミトコンドリア質量と膜電位の測定

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...