この研究は、NIH R00HL102154によってサポートされ、P智への血液学者賞のアメリカの社会ました。

このプロトコルに記載される実験は、ノースウェスタン大学施設内動物管理使用委員会によって定められたガイドラインおよび規制に従って行った。 培養液の調製<ol><li>フィブロネクチン溶液を調製する。ヒトフィブロネクチン（1mg）を1バイアルに1mlの水を追加します。撹拌せずに30分間組織培養フード内の溶液のままにしておきます。 20μg/ mlの最終濃度を作るために、PBS 50mlに全液体を移し、静かによく混ぜる。プレートを被覆する前に、フィブロネクチン溶液は新鮮行う（下記参照）。 </li><li>エポ培地（SCF培地）50mlのを準備します。新鮮作ったとき、表1に列挙した成分を組み合わせる。培地を1ヶ月間4℃で保存することができる。 </li><li>エポ含む培地50mlを準備します。新鮮作ったとき、表2に列挙した成分を組み合わせる。培地を1ヶ月間4℃で保存することができる。 </li></オール&gt; 2フィブロネクチンコートプレート<ol><li> 6ウェルプレート（または12ウェルプレートの各ウェルに0.5 ml）を各ウェルに1 mlのフィブロネクチン溶液を添加する。 1時間の組織培養フード内でプレートにしておきます。 </li><li>二回滅菌水でウェルを洗浄し、空気プレートを乾燥させます。 </li><li>プレートをラップし、4℃で低温室で保存してください。 </li></ol>胎児肝臓赤芽球の精製注：E13からE15時限妊娠マウス（C57BL / 6）に使用して胎児の肝臓。 E13.5肝臓はCFU-E前駆体の収量を最大にするために好ましい。時限妊娠マウスを得るためには、6〜8週齢の雄と雌のマウスは1ケージ（1つまたは2つの雌と通常1オス）に入れた。女性の膣のプラグは交尾が成功したかどうかを判断するために次の日の朝にチェックした。妊娠の最初の日は、プラグが検出された後の一日でした。 <ol><li>総胎児肝細胞の調製<ol><li>畝を生け贄に捧げるCO 2吸入と頸椎脱臼によってSE。消毒用70％エタノールで腹部をスプレーします。子宮を除去するために、はさみを解剖して、腹部を開きます。 1×PBS中で子宮を洗ってください。 </li><li>組織培養フード、オートクレーブのツールを使用して無菌条件下で胎児を解剖し、1×PBS中で洗浄します。 </li><li>胎児からの胎児の肝臓を解剖。別の鉗子で身体から離れ胎児肝臓（色はピンク）をゆっくりと引い1ピンセットで胎児のボディを持ってください。関連する線維性組織を除去するための鉗子で胎児の肝臓を清掃してください。 10％ウシ胎児血清を含む新鮮な1×PBS中にすべての胎児の肝臓を置きます。 </li><li>機械的ピペッティングにより胎児の肝臓を混乱させる。 </li><li> 50mlの円錐チューブに40μmのセルストレーナーを通して細胞懸濁液をフィルタリングします。 5分間800×gで細胞をペレット化。 </li><li> FBSの上清を吸引し、10％1mlのPBSで細胞を再懸濁（約8 E13.5胎児肝臓）1mlあたり。 </li></ol></li><li> TER119陰性赤芽球の精製<ol><li>氷上で15分間、ラットのIgG（0.2 mg / ml）を用いて細胞をブロックする。 </li><li>洗浄又は遠心分離せずに、細胞懸濁液（1μg/ ml）をビオチン化抗TER119抗体を加える。氷上で15分間インキュベートする。 </li><li> 5分間800×gで、10％FBS（1:10）と遠心分離機に1×PBS中で洗浄する。 10％FBSを含む1mlのPBSで細胞を再懸濁する。 </li><li>磁性粒子とコンジュゲート75μlのストレプトアビジンを加え、氷上で10分間インキュベートする。 </li><li> PBSで10％FBSを含む2.5ミリリットルにボリュームを追加します。ボトムチューブラウンド5ミリリットルのポリプロピレンに細胞懸濁液を転送します。 </li><li>磁気細胞選別装置内にチューブを挿入し、10分間インキュベートする。 TER119陽性細胞は、チューブの側面に付着する。アタッチされていないTER119負の赤芽球を懸濁液のままになります。 </li><li>ボトムチューブ新ラウンド5ミリリットルのポリプロピレンに細胞懸濁液を注ぐ。 </li><li><html>繰り返し、残留TER119陽性細胞を除去するために3.2.6と3.2.7を繰り返します。 </li><li> 5分間800×gで細胞をペレット化。上清を吸引除去する。 10％FBSを含む1mlのPBSで細胞を再懸濁し、トリパンブルーで生細胞を数える。 </li></ol></li></ol>ウイルスコードするcDNAまたはshRNAで4形質導入<ol><li> 3-5の精製TER119負の胎児肝細胞をプレー×10 5 /ウェルで12ウェルプレートをコーティングしたフィブロネクチン（異なるプレートを用いた場合に細胞数を調整する）である。 </li><li>ウイルス形質導入を容易にするためには10μg/ mlの最終濃度にポリブレンを加える。スピンは、37℃で1〜1.5時間、800×gで、cDNAまたはshRNAをコードするレンチウイルスまたはレトロウイルスで細胞を感染させる。 </li></ol>形質導入TER119陰性マウス胎児肝臓赤芽球の5。文化<ol><li>端末赤血球生成の初期段階での遺伝子の機能をテストするには（ 図1破線）。&quot;&gt; </ol.><ol><li><ol. li="">形質導入された遺伝子および前駆状態の維持の発現を可能にする12時間培養エポ培地で形質導入した細胞（SCF培地）。 </ol.></li><li> 5分間800×gで細胞を遠心。上清を吸引。 12ウェルプレートの各ウェルに培地を含有する1ミリリットルのEpo中で細胞を再懸濁する。 </li><li> 24または48時間培養後、さらなるアッセイのための細胞を採取する。 </li></ol></li><li>端末赤血球生成の後期段階でのテスト遺伝子機能（ 図1の実線）。 <ol><li>文化エポ含む培地中で、すぐに導入された細胞。 </li><li> 24または48時間培養後、さらなるアッセイのための細胞を採取する。 </li></ol></li></ol>フローサイトメトリー分析のために培養された胎児肝細胞の染色6。 <ol><li> 1×PBS中で細胞を洗浄し、再懸濁します。収穫フローサイトメトリー分析のために2×10 5個の培養細胞。 </li><li>培養する暗所で室温で20分間フルオロフォア結合抗体を有する細胞。分化をテストするには、APC結合抗CD71およびPE結合抗TER119を使用しています。摘出をテストするには、他の抗体に加えて、ヘキスト33342染色を使用しています。 </li><li> 1×PBSで細胞を洗浄する。死細胞を染色し、分析から除外するために、1％FBSおよびヨウ化プロピジウム（PI）（1μg/ ml）を含む0.5 mlのPBS中に細胞を再懸濁。 </li><li>流れcytrometric分析を実施する。フローサイトメーターの調整と補償のために最初に単一の色に染色し、染色していない対照細胞を分析します。 </li></ol>

<ol><li>Hattangadi, S. M., Wong, P., Zhang, L., Flygare, J., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+stem+cell+to+red+cell%3A+regulation+of+erythropoiesis+at+multiple+levels+by+multiple+proteins%2C+RNAs%2C+and+chromatin+modifications%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">From stem cell to red cell: regulation of erythropoiesis at multiple levels by multiple proteins, RNAs, and chromatin modifications.</a> Blood. 118 (24), 6258-6268 (2011).</li>
<li>Richmond, T. D., Chohan, M., Barber, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Turning+cells+red%3A+signal+transduction+mediated+by+erythropoietin%5BTitle%5D)+AND+%22Trends+in+cell+biology%22%5BJournal%5D)">Turning cells red: signal transduction mediated by erythropoietin.</a> Trends in cell biology. 15 (3), 146-155 (2005).</li>
<li>Eshghi, S., Vogelezang, M. G., Hynes, R. O., Griffith, L. G., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alpha4beta1+integrin+and+erythropoietin+mediate+temporally+distinct+steps+in+erythropoiesis%3A+integrins+in+red+cell+development%5BTitle%5D)+AND+%22The+Journal+of+cell+biology%22%5BJournal%5D)">Alpha4beta1 integrin and erythropoietin mediate temporally distinct steps in erythropoiesis: integrins in red cell development.</a> The Journal of cell biology. 177 (5), 871-880 (2007).</li>
<li>Koury, M. J., Bondurant, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maintenance+by+erythropoietin+of+viability+and+maturation+of+murine+erythroid+precursor+cells%5BTitle%5D)+AND+%22Journal+of+cellular+physiology%22%5BJournal%5D)">Maintenance by erythropoietin of viability and maturation of murine erythroid precursor cells.</a> Journal of cellular physiology. 137 (1), 65-74 (1988).</li>
<li>Ji, P., Yeh, V., Ramirez, T., Murata-Hori, M., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histone+deacetylase+2+is+required+for+chromatin+condensation+and+subsequent+enucleation+of+cultured+mouse+fetal+erythroblasts%5BTitle%5D)+AND+%22Haematologica%22%5BJournal%5D)">Histone deacetylase 2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal erythroblasts.</a> Haematologica. 95 (12), 2013-2021 (2010).</li>
<li>Dumitriu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sox6+cell-autonomously+stimulates+erythroid+cell+survival%2C+proliferation%2C+and+terminal+maturation+and+is+thereby+an+important+enhancer+of+definitive+erythropoiesis+during+mouse+development%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Sox6 cell-autonomously stimulates erythroid cell survival, proliferation, and terminal maturation and is thereby an important enhancer of definitive erythropoiesis during mouse development.</a> Blood. 108 (4), 1198-1207 (2006).</li>
<li>Rector, K., Liu, Y., Van Zant, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comprehensive+hematopoietic+stem+cell+isolation+methods%5BTitle%5D)+AND+%22Methods+in+molecular+biology%22%5BJournal%5D)">Comprehensive hematopoietic stem cell isolation methods.</a> Methods in molecular biology. 976, 1-15 (2013).</li>
<li>Rossi, L., Challen, G. A., Sirin, O., Lin, K. K., Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hematopoietic+stem+cell+characterization+and+isolation%5BTitle%5D)+AND+%22Methods+in+molecular+biology%22%5BJournal%5D)">Hematopoietic stem cell characterization and isolation.</a> Methods in molecular biology. 750, 47-59 (2011).</li>
<li>Choi, H. S., Lee, E. M., Kim, H. O., Park, M. I., Baek, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autonomous+control+of+terminal+erythropoiesis+via+physical+interactions+among+erythroid+cells%5BTitle%5D)+AND+%22Stem+cell+research%22%5BJournal%5D)">Autonomous control of terminal erythropoiesis via physical interactions among erythroid cells.</a> Stem cell research. 10 (3), 442-453 (2013).</li>
<li>Bhatia, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Short-chain+fatty+acid-mediated+effects+on+erythropoiesis+in+primary+definitive+erythroid+cells%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Short-chain fatty acid-mediated effects on erythropoiesis in primary definitive erythroid cells.</a> Blood. 113 (25), 6440-6448 (2009).</li>
<li>Zhang, J., Socolovsky, M., Gross, A. W., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+Ras+signaling+in+erythroid+differentiation+of+mouse+fetal+liver+cells%3A+functional+analysis+by+a+flow+cytometry-based+novel+culture+system%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry-based novel culture system.</a> Blood. 102 (12), 3938-3946 (2003).</li>
<li>Socolovsky, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ineffective+erythropoiesis+in+Stat5a%28-%2F-%295b%28-%2F-%29+mice+due+to+decreased+survival+of+early+erythroblasts%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Ineffective erythropoiesis in Stat5a(-/-)5b(-/-) mice due to decreased survival of early erythroblasts.</a> Blood. 98 (12), 3261-3273 (2001).</li>
<li>Ji, P., Jayapal, S. R., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enucleation+of+cultured+mouse+fetal+erythroblasts+requires+Rac+GTPases+and+mDia2%5BTitle%5D)+AND+%22Nature+cell+biology%22%5BJournal%5D)">Enucleation of cultured mouse fetal erythroblasts requires Rac GTPases and mDia2.</a> Nature cell biology. 10 (3), 314-321 (2008).</li>
<li>Chida, D., Miura, O., Yoshimura, A., Miyajima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+cytokine+signaling+molecules+in+erythroid+differentiation+of+mouse+fetal+liver+hematopoietic+cells%3A+functional+analysis+of+signaling+molecules+by+retrovirus-mediated+expression%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Role of cytokine signaling molecules in erythroid differentiation of mouse fetal liver hematopoietic cells: functional analysis of signaling molecules by retrovirus-mediated expression.</a> Blood. 93 (5), 1567-1578 (1999).</li>
<li>Patel, V. P., Lodish, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+fibronectin+matrix+is+required+for+differentiation+of+murine+erythroleukemia+cells+into+reticulocytes%5BTitle%5D)+AND+%22The+Journal+of+cell+biology%22%5BJournal%5D)">A fibronectin matrix is required for differentiation of murine erythroleukemia cells into reticulocytes.</a> The Journal of cell biology. 105 (6 Pt 2), 3105-3118 (1987).</li>
<li>Udupa, K. B., Lipschitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endotoxin-induced+suppression+of+erythropoiesis%3A+the+role+of+erythropoietin+and+a+heme+synthesis+stimulating+factor%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Endotoxin-induced suppression of erythropoiesis: the role of erythropoietin and a heme synthesis stimulating factor.</a> Blood. 59 (6), 1267-1271 (1982).</li>
<li>Koury, M. J., Sawyer, S. T., Brandt, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+insights+into+erythropoiesis%5BTitle%5D)+AND+%22Current+opinion+in+hematology%22%5BJournal%5D)">New insights into erythropoiesis.</a> Current opinion in hematology. 9 (2), 93-100 (2002).</li>
</ol>

著者は、彼らが競合する金融利害がないことを宣言します。

ここでは、時系列的に、マウス胎児肝臓端末赤血球生成を分析する独自のシステムを提示する。異なる培養条件の適用を通じて、首尾よく初期および後期段階での端末赤血球生成を解剖した。これは、複数の機能を有する遺伝子のメカニズムを決定するために特に重要である。例えば、RacのGTPアーゼは、端末赤血球生成の各段階において重要な役割を果たしている。端末赤血球生成に影響を?...

赤血球生成は、赤血球を成熟多能造血幹細胞の分化の過程である。この段階的なプロセスは、コミットされた赤血球バースト形成単位（BFU-ES）、急速に分裂赤血球コロニー形成単位（CFU-ES）、及び形態学的に認識可能な赤芽球1,2の形成を含む。 CFU-Eの前駆細胞からの端末赤血球生成は、シーケンシャルエリスロポエチン依存性および非依存性のステージ2,3を伴う。端末赤血球形成の初期段階では、エリスロポエチン（EPO）は、細胞表面上の受容体に結合し、細胞アポトーシスを防止し、迅速な細胞分裂および遺伝子発現1,4を促進する下流のシグナル伝達経路のシリーズを誘導する。端末赤血球生成の後期段階では、赤芽球は、高度に凝縮した核5の端子分裂停止、クロマチンおよび核の凝縮、および押出を受ける。 端末の理解赤血球生成が大幅にin vitroおよびin vivoで 6-9マウスモデルにおけるいくつかの使用の成功によるところが大きい、これ過去数十年で改善されています。これらのモデルの中でも、マウス胎児肝芽球のインビトロ培養中の細胞の精製、速い増殖および分化の容易さを含む多くの利点を提供し、人間の赤血球生成10,11に近い模倣。このシステムでは、マウス胎児肝臓からの赤血球前駆細胞の多数が容易にTER119の単一ステップ精製（成熟赤血球系細胞のマーカー）は、負の赤芽球を単離することができる。赤芽球の二日間の培養の間、これらの細胞の分化は、トランスフェリン受容体（CD71）の表面発現およびTER119抗原12に基づいて、フローサイトメトリー分析によってモニターすることができる。また、末端分化赤芽球の除核は、DNAメーカー（ヘキスト33342）によって検出することができる13。さらに、精製された前駆細胞は、遺伝的に赤血球生成11,13,14上の遺伝子発現の機能の機構研究を容易にする、目的の遺伝子のcDNAまたは小ヘアピンRNA（shRNAは）の外因性発現によって改変することができる。 それは、端末赤血球形成の異なる段階での遺伝子機能を特徴付けることは困難であるので、一方で、高速細胞増殖速度は、両刃の剣であることができる。ほとんどの場合、それは、cDNAまたはshRNAが発現した時点で以降の端末赤血球形成の初期段階における特定の遺伝子の機能は、細胞が既に初期段階に合格したかどうかを決定することは困難である。この問題を解決するために、私たちは、端末赤血球生成の初期および後期段階を詳細に分析するために独自のシステムを開発しました。端末赤血球形成の初期段階では、遺伝的に改変されTER119負赤芽球は、Epoを含まない培地中で培養したが、幹細胞因子（SCF）、IL-6およびFLT3を含むリガンドは、それらの前駆状態を維持し、数13に形質導入されたcDNAまたはshRNAを可能にする。細胞は、細胞の増殖および分化を誘導するために12時間後にEpoを含む培地に変更した。細胞が分化し始めたときにこのように、形質導入されたcDNAまたはshRNAは、既に発現した。端末赤血球生成の後期段階では、TER119負の赤芽球は、すぐ​​に導入後エポ含む培地で培養した。従って、一つの端末赤血球形成の後期段階において、目的の遺伝子の機能を分析することができる。要約すると、本システムの広範な適用は、端末赤血球形成の異なる段階での遺伝子機能を分析に役立つだろう。

<table><tbody><tr><td>Iscove&#039;s Modified Dulbecco&#039;s MediumIMDM (IMDM)</td><td>Gibco</td><td>12440-053</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>GEMINI Bio-product</td><td>700-102P</td><td></td></tr><tr><td>Penicillin-Streptomycin solution</td><td>Hyclone</td><td>SV30010</td><td>100x</td></tr><tr><td>L-Glutamine</td><td>Hyclone</td><td>SH30034.01</td><td>200 mM</td></tr><tr><td>Mouse FIT3 Ligand (Flt-3L)</td><td>BD Biosciences&amp;nbsp;</td><td>14-8001-80</td><td></td></tr><tr><td>Mouse Recombinant Interleukin-6 (IL-6)&amp;nbsp;</td><td>GEMINI Bio-product</td><td>300-327P</td><td></td></tr><tr><td>Fibronectin</td><td>BD Biosciences&amp;nbsp;</td><td>354008</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>STEMCELL TECH</td><td>9300</td><td>10% stock in IMDM</td></tr><tr><td>Erythropoietin(Epo)</td><td>PROCRIT</td><td>NOC 59676-303-00</td><td>3,000 U/ml stock</td></tr><tr><td>Streptavidin particles Plus</td><td>BD Pharmingen</td><td>557812</td><td></td></tr><tr><td>EasySep Magnet</td><td>STEMCELL TECH</td><td>18000</td><td></td></tr><tr><td>Polypropylene Round-Bottom Tube, 5 ml</td><td>FALCON</td><td>352063</td><td></td></tr></tbody></table>

mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis

赤血球生成が急速に形成単位赤血球コロニー（CFU-ES）を分周して、その後、コミット赤血球バースト形成単位（BFU-ES）で始まる動的なプロセスを伴う。 CFU-ESの後、細胞を形態学的に認識され、一般的には、端末赤芽球と呼ばれる。端末赤血球生成の研究のための課題の一つは、時系列的に遺伝子の機能を分析するための実験的アプローチの欠如である。このプロトコルでは、端末赤血球形成の初期及び後期段階での遺伝子機能を決定するためのユニークな戦略を記載している。このシステムでは、マウス胎児肝臓TER119（成熟赤血球細胞マーカー）負の赤芽球を精製し、目的の遺伝子のcDNAや小さなヘアピンRNA（shRNAを）の外因性発現で形質導入。細胞は、外因性cDNAまたはshRNAを可能にしながら、12時間それらの前駆段階を維持するためにエリスロポエチン（EPO）以外の成長因子を含む培地で培養し、その後発現させた。外因性遺伝物質がすでに発現させながら、細胞は、細胞分化および増殖を誘導するために12時間後のEpo培地に変更した。このプロトコルは、端末赤血球生成の初期段階で遺伝子機能の解析を容易にします。後期端末赤血球形成を研究するために、細胞を直ちに形質導入​​後のEpo培地中で培養した。このように、細胞は、既に形質導入​​された遺伝物質を発現した端末赤血球形成の後期に分化させた。私たちは、端末赤血球生成のさまざまな段階での詳細な遺伝子機能を理解する助けとなるこの戦略の一般的な適用を推奨します。

図1は、実験的な戦略の概要を説明します。プロトコルは、端末赤血球生成の初期および後期段階でのシグナル伝達分子の機能を標的とするための2つの独立した条件で構成されています。 TER119陰性胎児肝芽球は、E13.5マウス胎児から精製した。胎児肝臓赤血球系細胞の前および精製後のフローサイトメトリー分析により、精製が（ 図2Aおよび2B）効率的?...

Watch this Scientific Journal Video about Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis at JoVE.com

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

私たちは、端末赤血球生成の初期および後期段階を解剖インビトロマウス胎児肝臓赤芽球培養系を提示。このシステムは、異なる発生段階における特定の遺伝子の機能解析を容易にします。

マウス胎児肝臓培養システムは、ターミナル赤血球の初期および後期段階での標的遺伝子の機能を分析する

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

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A Hierarchical Stem-Cell System: Blood Cell Formation

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15.0K ビュー.  Northwestern University. 私たちは、端末赤血球生成の初期および後期段階を解剖インビトロマウス胎児肝臓赤芽球培養系を提示。このシステムは、異なる発生段階における特定の遺伝子の機能解析を容易にします。 

ビデオ: Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

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 ...

発生生物学

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

フローサイトメトリーによるマウス胚における赤血球系前駆細胞およびその子孫の同定

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

リネージュの赤芽球系前駆細胞に成体マウス線維芽細胞のリプログラミングを直接します。

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

フローサイトメトリーによるマウス組織からのバースト形成ユニットおよびコロニー形成ユニット赤血球前駆細胞の同定と単離(英語)

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

CD71/TER119フローサイトメトリーアッセイを用いてマウス赤血球前駆細胞の同定と解析

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

生物学

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 ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

マルチスペクトルイメージングフローサイトメトリーによる除核イベントにおけるマウス赤芽球亜集団エンリッチの同定

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

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 ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

マウス肝臓における細胞型特異的遺伝子発現プロファイリング

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

<meta charset="utf8"></head><body>臍帯血および骨髄からのHSPCを使用した赤血球生成のモデリング

A Comprehensive Pipeline to Assess the Efficiency of Human Erythropoiesis In Vitro and Ex Vivo

Erythropoiesis, a remarkably dynamic and efficient process responsible for generating the daily quota of red blood cells (approximately 280 &plusmn; 20 billion cells per day), is crucial for maintaining individual health. Any disruption in this pathway can have significant consequences, leading to health issues. According to the World Health Organization, an estimated 25% of the global population presents symptoms of anemia. This protocol describes how to generate human erythroid cells both in vitro using hematopoietic stem and progenitor cells (HSPCs) from sources such as umbilical cord blood (UCB) or blood taken from healthy donors and ex vivo with HSPCs isolated from patients' bone marrow. Using genetic approach, genes of interest can be modulated in HSPCs, and their impact on erythropoiesis can be monitored at various stages of the differentiation process. This method allows for the screening of compounds perturbing, enhancing, or rescuing the capacity of HSPCs to differentiate into mature erythroid cells and to investigate the role of genes of interest during the erythroid differentiation process.

<meta charset="utf8"></head><body>著者スポットライト:赤血球生成研究の進歩 - 骨髄異形成症候群における造血幹細胞機能の評価のための簡素化されたパイプライン

Modeling erythropoiesis provides a valuable chance to comprehend the biological significance of actors in this process and to evaluate novel therapeutic approaches that may alleviate differentiation defects. Here, we describe a simple and reliable method to efficiently differentiate CD34+ hematopoietic stem and progenitor cells ex vivo.

ヒト赤血球生成の効率をin vitroおよびex vivoで評価するための包括的なパイプライン

Erythropoiesis, a remarkably dynamic and efficient process responsible for generating the daily quota of red blood cells (approximately 280 &plusmn; 20 ...