Name: suppression of pro-fibrotic signaling potentiates factor-mediated reprogramming of mouse embryonic fibroblasts into induced cardiomyocytes
Uploaded: 2018-06-03T14:00:00
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この研究は、ベッチャー財団のウェッブ ワーミング生物医学研究プログラム、アメリカ心臓協会の科学者開発補助 (13SDG17400031) コロラド大学部門医学優れた初期のキャリアからの資金によって支えられました。学者プログラムは、コロラド州部の循環器バーロー Nyle 基金、大学と NIH (カンザス) に R01HL133230。A.S.R は、線維症研究 & 翻訳 (CFReT) の NIH/NCATS コロラド CTSA 許可番号 TL1TR001081 とコロラド大学コンソーシアムから博士フェローシップによって支持されました。本研究は、がんセンター助成金 (P30CA046934)、皮膚疾患研究コア助成金 (P30AR057212)、コロラド大学アンシュッツ メディカル キャンパスで流れ Cytometry コアによっても支えられました。

動物を必要とするすべての実験は、動物介護制度と UC デンバー アンシュッツ医療キャンパス利用委員会によって承認されました。
1. MEFs の分離
<ol><li>E13 の C57BL/6 妊娠マウスを購入します。一晩出荷。</li>
	<li>承認された IACUC プロトコルによると母親を安楽死させる (ex: 頚部転位続いて ~1.3 L/min CO2動物が死んだが表示されるまで)</li>
	<li>70% エタノールで母親を?...

<ol>
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G., Braunwald, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tale+of+coronary+artery+disease+and+myocardial+infarction.">A tale of coronary artery disease and myocardial infarction.</a> N Engl J Med. 366 (1), 54-63 (2012).</li><li>Packer, M., 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=Effect+of+carvedilol+on+the+morbidity+of+patients+with+severe+chronic+heart+failure:+results+of+the+carvedilol+prospective+randomized+cumulative+survival+(COPERNICUS)+study.">Effect of carvedilol on the morbidity of patients with severe chronic heart failure: results of the carvedilol prospective randomized cumulative survival (COPERNICUS) study.</a> Circulation. 106 (17), 2194-2199 (2002).</li><li>The CAPRICORN Investigators. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+carvedilol+on+outcome+after+myocardial+infarction+in+patients+with+left-ventricular+dysfunction:+the+CAPRICORN+randomized+trial.">Effect of carvedilol on outcome after myocardial infarction in patients with left-ventricular dysfunction: the CAPRICORN randomized trial.</a> The Lancet. 357 (9266), 1385-1390 (2001).</li><li>Marangou, J., Paul, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+attitudes+on+cardiac+devices+in+heart+failure:+a+review.">Current attitudes on cardiac devices in heart failure: a review.</a> Clin Ther. 37 (10), 2206-2214 (2015).</li><li>Xin, M., Olson, E. N., Bassel-Duby, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mending+broken+hearts:+cardiac+development+as+a+basis+for+adult+heart+regeneration+and+repair.">Mending broken hearts: cardiac development as a basis for adult heart regeneration and repair.</a> Nat Rev Mol Cell Bio. 14 (8), 529-541 (2013).</li><li>Wollert, K. C., 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=Intracoronary+autologous+bone-marrow+cell+transfer+after+myocardial+infarction:+the+BOOST+randomized+controlled+clinical+trial.">Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomized controlled clinical trial.</a> Lancet. 364 (9429), 141-148 (2004).</li><li>Schachinger, V., 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=Intracoronary+bone+marrow-derived+progenitor+cells+in+acute+myocardial+infarction.">Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction.</a> N. Engl. J. Med. 355 (12), 1210-1221 (2006).</li><li>Huikuri, H. 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Heart J. 29 (22), 2723-2732 (2008).</li><li>Janssens, S., 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=Autologous+bone+marrow-derived+stem-cell+transfer+in+patients+with+ST-segment+elevation+myocardial+infarction:+double-blind,+randomised+controlled+trial.">Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlled trial.</a> Lancet. 367 (9505), 113-121 (2006).</li><li>Lunde, K., 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=Intracoronary+injection+of+mononuclear+bone+marrow+cells+in+acute+myocardial+infarction.">Intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction.</a> N. Engl. J. Med. 355 (12), 1199-1209 (2006).</li><li>Hirsch, A., 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=Intracoronary+infusion+of+autologous+mononuclear+bone+marrow+cells+or+peripheral+mononuclear+blood+cells+after+primary+percutaneous+coronary+intervention:+rationale+and+design+of+the+HEBE+trial+-+a+prospective,+multicenter,+randomized+trial.">Intracoronary infusion of autologous mononuclear bone marrow cells or peripheral mononuclear blood cells after primary percutaneous coronary intervention: rationale and design of the HEBE trial - a prospective, multicenter, randomized trial.</a> Am. Heart J. 152 (3), 434-441 (2006).</li><li>Hirsch, A., 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=Intracoronary+infusion+of+mononuclear+cells+from+bone+marrow+or+peripheral+blood+compared+with+standard+therapy+in+patients+after+acute+myocardial+infarction+treated+by+primary+percutaneous+coronary+intervention:+results+of+the+randomized+controlled+HEBE+trial.">Intracoronary infusion of mononuclear cells from bone marrow or peripheral blood compared with standard therapy in patients after acute myocardial infarction treated by primary percutaneous coronary intervention: results of the randomized controlled HEBE trial.</a> Eur. Heart J. 32 (14), 1736-1747 (2011).</li><li>Karantalis, V., 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=Autologous+mesenchymal+stem+cells+produce+concordant+improvements+in+regional+function,+tissue+perfusion,+and+fibrotic+burden+when+administered+to+patients+undergoing+coronary+artery+bypass+grafting:+The+Prospective+Randomized+Study+of+Mesenchymal+Stem+Cell+Therapy+in+Patients+Undergoing+Cardiac+Surgery+(PROMETHEUS)+trial.">Autologous mesenchymal stem cells produce concordant improvements in regional function, tissue perfusion, and fibrotic burden when administered to patients undergoing coronary artery bypass grafting: The Prospective Randomized Study of Mesenchymal Stem Cell Therapy in Patients Undergoing Cardiac Surgery (PROMETHEUS) trial.</a> Circ Res. 114 (8), 1302-1310 (2014).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li><li>Lian, X., 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=Robust+cardiomyocyte+differentiation+from+human+pluripotent+stem+cells+via+temporal+modulation+of+canonical+Wnt+signaling.">Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.</a> Proc Natl Acad Sci. 109 (27), E1848-E1857 (2012).</li><li>Loh, K. M., 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=Mapping+the+Pairwise+Choices+Leading+from+Pluripotency+to+Human+Bone,+Heart,+and+Other+Mesoderm+Cell+Types.">Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types.</a> Cell. 166 (2), 451-467 (2016).</li><li>Yoshida, Y., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPS+cells:+A+source+of+cardiac+regeneration.">iPS cells: A source of cardiac regeneration.</a> J. Mol. Cell. 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本研究では、直接初期化因子プロ線維性シグナル伝達経路の抑制と組み合わせる GHMT2m の配信を介して機能 iCMs に線維芽細胞を再プログラムする高効率戦略について説明します。フローサイトメトリー、蛍光イメージング、カルシウム イメージング、およびこのプロトコルでセルの大半を示す細胞数を打つことを使用して成功の書き換えを受けるし、CM リネージュ運命を採用します。我々 は...

虚血性心疾患は、アメリカ合衆国1死の主要な原因です。およそ 800,000 のアメリカ人の経験最初または再発性心筋梗塞 (MI)1年につき。MI、次は、心筋 (CMs) と活性化心臓線維芽細胞による心筋の線維化の死は心臓機能2,3を損ないます。MI の後心不全の進行は主大人 CMs4、5の悪い再生能力により元に戻すこと。現在臨床治療が病気の進行を遅らせる、将来心臓イベント6,7,8,9のリスクを減少させる治療はできないのための病気の進行を逆します。CMs 心筋硬塞後10を再生成します。マイル失意のうち、次の患者を治療するために浮上している新規細胞治療、MI をこれまで続く心臓に幹細胞を提供する臨床試験は決定的でない再生潜在的な11,12,を示されています。13,14,15,16,17,18。
最初高橋 & 山中で示した 4 つの転写因子の過剰発現による線維芽細胞からひと由来の誘導多能性幹細胞 (hiPSCs) の生成は、細胞療法19で新たなブレークスルーにドアを開けた。これらの細胞は、すべて 3 つの胚葉19に区別できる、CMs の大規模な番号を生成するためのいくつかの非常に効率的な方法は、以前20,21を示されています。派生した CMs (腰-CMs) 心筋を勉強する強力なプラットフォームを提供しています、心臓の損傷を修復するための重要な含意があるかもしれない。腰 CMs 現在奇形腫形成22, 懸念のため並進ハードルに直面し、その未熟な性質があります pro 不整脈23。直接他のセルタイプに線維芽細胞をリプログラミングの興味を引き起こした hiPSCs に線維芽細胞をプログラムし直します。家田らは、低効率24どまる Tbx5 (GMT)、Mef2c、GATA4 心臓系統に直接再プログラム化により、線維芽細胞で過剰発現を示した。リプログラミング効率 Hand2 (GHMT)25の付加と改善されました。これらの初期の研究から多くの出版物を示していることは26,27,28,29, 要因追加転写リプログラミング因子カクテルを変更34は改善につながる小さな分子やマイクロ Rna32,33, クロマチン修飾子30,31リプログラミング効率および/または誘導された心筋細胞のような細胞 (iCMs の成熟).
ここでは、高効率でマウス萌芽期の繊維芽細胞 (MEFs) から iCMs を生成する詳細なプロトコルを提供します。以前示したカクテル GHMT ミール 1 とミール-133 (GHMT2m) の追加で大幅に改善され、さらに向上、プロ線維増殖因子 β (TGF-β) シグナリングを変換を含むシグナルまたは Rho 関連蛋白質キナーゼ (ロック) シグナル伝達経路が抑制35です。このプロトコルを使用して、ことを示す細胞の約 60% エクスプレス心臓トロポニン T (cTnT)、50% 約表現 α-アクチニン暴行細胞数が多いが、早ければ次の要因と治療をリプログラミングの伝達日 11 観察できます。TGF-β と I 型受容体阻害剤 A-83-01 です。さらに、これらの iCMs はギャップ結合蛋白質コネキシン 43 を含むを表し、自発収縮とカルシウム濃度を示します。このマークが以前の研究と比較して効率をリプログラミングの改善は心後梗塞に残っている内因性の細胞集団から CMs を再生成する可能性を示しています。

<table border="0" cellpadding="0" cellspacing="0" width="1326">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">C57BL/6 Mice</td>
			<td style="width:197px;">Charles River&#39;s Laboratory</td>
			<td style="width:111px;">027</td>
			<td style="width:667px;">For MEF isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum E (PE) Cells</td>
			<td>Cell Biolabs, INC</td>
			<td>RV-101</td>
			<td>For retrovirus production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM High Glucose</td>
			<td>Gibco</td>
			<td>SH30022.FS</td>
			<td>Component of iCM, PE, and Growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medium 199</td>
			<td>Life Technologies</td>
			<td>11150-059</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Gemini</td>
			<td>100106</td>
			<td>Component of iCM, PE, and Growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donor Horse Serum</td>
			<td>Gemini</td>
			<td>100508 500</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">MEM Essential Amino Acids, 50X</td>
			<td>Life Technologies</td>
			<td>11130051</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Sodium Pyruvate Solution, 100X</td>
			<td>Life Technologies</td>
			<td>11360070</td>
			<td>Component of iCM media and for calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">MEM Non-Essential Amino Acids, 100X</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">MEM Vitamin Solution, 100X</td>
			<td>Life Technologies</td>
			<td>11120-052</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Insulin-Transferrin-Selenium</td>
			<td>Gibco</td>
			<td>41400045</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Penicilin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Component of iCM, PE, and Growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">GlutaMAX (L-Glutamine Supplement)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>Component of iCM, PE, and Growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blasticidin-HCl</td>
			<td>Life Technologies</td>
			<td>A11139-03</td>
			<td>Component of PE media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Puromycin dihydrochloride</td>
			<td>Life Technologies</td>
			<td>A11138-03</td>
			<td>Component of PE media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">0.25% Trypsin/EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>For detaching cells from culture dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A-83-01</td>
			<td>R&#38;D Systems - Tocris</td>
			<td>2939/10</td>
			<td>Treat cells to inhibit TGF-&#946; signaling - promotes high efficiecy reprogramming. Use at 0.5 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Thermo Scientific</td>
			<td>85190</td>
			<td>For dilution and storage of A-83-01 and component of Freeze Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SureCoat</td>
			<td>Cellutron</td>
			<td>SC-9035</td>
			<td>For coating dishes to plate MEFs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FuGENE 6 Transfection Reagent</td>
			<td>Promega</td>
			<td>E2692</td>
			<td>Transfection Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM Reduced Serum Media</td>
			<td>Gibco</td>
			<td>11058-021</td>
			<td>Transfection Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X Myc-GATA4</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X Myc-Hand2</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X Myc-Mef2c</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X Myc-Tbx5</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X miR-1</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X miR-133</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X GFP</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polybrene (Hexadimethrine bromide)</td>
			<td>Sigma</td>
			<td>H9268-5G</td>
			<td>For viral induction. Use at a concentration of 6 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Vacuum Filter + bottles (0.22 &#181;m pores)</td>
			<td>Nalgene</td>
			<td>&#160;569-0020&#160;</td>
			<td>For filtering media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringes</td>
			<td>Bd Vacutainer Labware&#160;</td>
			<td>309654</td>
			<td>For viral filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 &#181;m Filters</td>
			<td>Celltreat</td>
			<td>229749</td>
			<td>For viral filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 &#181;m cell strainers</td>
			<td>Falcon&#160;</td>
			<td>352350</td>
			<td>For MEF isolation and Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Cytofix/Cytoperm Solution</td>
			<td>BD</td>
			<td>554722</td>
			<td>For fixation and permeabilization of cells for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">perm/wash buffer&#160;</td>
			<td>BD</td>
			<td>554723</td>
			<td>For washing cells for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS 1X</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td>For washing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>VWR</td>
			<td>0332-100g</td>
			<td>For flow cytometry and calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Serum</td>
			<td>Sigma&#160;</td>
			<td>G9023</td>
			<td>For blocking cells for Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey Serum</td>
			<td>Sigma</td>
			<td>D9663-10mg</td>
			<td>For blocking cells for Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Troponin T</td>
			<td>Thermo Scientific</td>
			<td>ms-295-p</td>
			<td>1:400 IF, 1:200 Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse &#945;-actinin</td>
			<td>Sigma</td>
			<td>A7811L</td>
			<td>1:400 IF, 1:200 Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit Connexin 43</td>
			<td>Sigma&#160;</td>
			<td>C6219</td>
			<td>1:400 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit Troponin I</td>
			<td>PhosphoSolutions</td>
			<td>2010-TNI</td>
			<td>1:400 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst</td>
			<td>Life Technologies</td>
			<td>62249</td>
			<td>1:10000 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 488, rabbit</td>
			<td>Life Technologies</td>
			<td>A-11034</td>
			<td>1:800 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 555, mouse</td>
			<td>Life Technologies</td>
			<td>A-21422</td>
			<td>1:800 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 647, mouse</td>
			<td>Life Technologies</td>
			<td>A-31571</td>
			<td>1:200 Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">27-color ZE5 Flow Cytometer&#160;</td>
			<td>Bio-RAD</td>
			<td></td>
			<td>For FACS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>sigma</td>
			<td>P6148-500mg</td>
			<td>For fixing cells for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Promega</td>
			<td>H5142</td>
			<td>For permeabilization of cells for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EVOS&#8482; FL Color Imaging System</td>
			<td>Thermo Scientific</td>
			<td>AMEFC4300</td>
			<td>For IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>RPI</td>
			<td>S23020-5000</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>VWR</td>
			<td>395</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">CaCl2</td>
			<td>Fisher</td>
			<td>C614-500</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2</td>
			<td>VWR</td>
			<td>97061-352</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glucose</td>
			<td>sigma</td>
			<td>G7528-250g</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>sigma</td>
			<td>H4034-500g</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fura-2 AM</td>
			<td>Life Technologies</td>
			<td>F1221</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluronic F-127</td>
			<td>Sigma</td>
			<td>P2443-250g</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nifedipine</td>
			<td>Sigma</td>
			<td>N7634-1G</td>
			<td>For disruption calcium transients in iCMs - use at 10 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoproterenol</td>
			<td>sigma</td>
			<td>I6504-1g</td>
			<td>For increasing number of calcium transients in iCMs - use at 1-2 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Marianas Spinning Disk Confocal microscope</td>
			<td>3i</td>
			<td></td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol</td>
			<td>Decon Laboratories</td>
			<td>2801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bleach</td>
			<td>Clorox</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical tubes</td>
			<td>GREINER BIO-ONE</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL conical tubes</td>
			<td>GREINER BIO-ONE</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 cm cell culture dishes</td>
			<td>Falcon</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 cm cell culture dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm cell culture dishes</td>
			<td>GREINER BIO-ONE</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well cell culture plates</td>
			<td>GREINER BIO-ONE</td>
			<td>657160&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12 well cell culture plates</td>
			<td>GREINER BIO-ONE</td>
			<td>665180&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well cell culture plates</td>
			<td>GREINER BIO-ONE</td>
			<td>662160&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

suppression of pro-fibrotic signaling potentiates factor-mediated reprogramming of mouse embryonic fibroblasts into induced cardiomyocytes

別に 1 つの体細胞のタイプのトランス分化には、モデルおよび人間の病気の治療に莫大な可能性があります。以前研究示されている、マウス胚は、真皮、そして心臓線維芽細胞を誘導心筋細胞様細胞 (iCMs) に書き換えることができます GATA4、Hand2 Mef2c など心原性転写因子の過剰発現を介して、Tbx5の in vitroとin vivoの両方。ただし、これらの以前の研究は、比較的低効率を示しています。心臓機能障害を復元するために iCMs の成熟と効率を向上させる心臓のリプログラミングを支配するメカニズムを解明する必要があります。
以前、リプログラミング効率を劇的にプロ線維のシグナル伝達の阻害に増加を示した。ここでは、リプログラミング効率 60% を達成する方法を詳しく説明します。また、フローサイトメトリー、蛍光イメージングは、リプログラミング効率とプログラムし直された線維芽細胞の成熟を定量化するカルシウム イメージングなどいくつかの方法をについて説明します。ここで詳細なプロトコルを使用して、心臓のリプログラミングの正と負のレギュレータを決定する解明を引き受けることができます。これらの研究は、リプログラミング効率と人間の心臓病を治療する細胞治療法につながる可能性の成熟を促進するためにターゲットすることができますシグナリング経路を識別できます。

約 70% と心筋トロポニン T を発現する細胞の心筋 α-アクチニン、日 9 にフローサイトメトリーによる定量化を発現する細胞の約 55% の iCMs 上記及び図 1Bリプログラミングの戦略を使用して、生成しました。次の GHMT2m の伝達 (図 2 aとB)。さらに、細胞の大部分はエクスプレス心臓トロポニン T、トロポニンは心筋...

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Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

機能的な心筋細胞発現 Tbx5、ミール 1 とミール 133 Mef2c、Hand2 GATA4 (GHMT2m) と一緒に TGF-β シグナルの阻害を介してプライマリ胚性線維芽細胞を再プログラムする手法をご紹介します。我々 のプロトコル 60% までの 7 日間後の伝達も早く拍動心筋細胞を生成する効率。

プロ線維性シグナル伝達の抑制を促進する誘導心筋細胞マウス胚性線維芽細胞のリプログラミング因子を介した

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

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Developmental Biology

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

神経の分析のための成体マウスのV-SVZにおける細胞の安定的かつ効率的な遺伝子改変幹細胞自律と非自律効果

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

GATA4、MEF2C及びTBX5の最適な比率を表現ポリシストロン構築物を用いて誘導心筋細胞の改善された世代

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

栄養膜幹細胞へのマウス胚性線維芽細胞の直接変換するためのプロトコル

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

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

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 ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

マウスの破骨細胞前駆体におけるNotchシグナル伝達の刺激

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

マウス胚線維芽細胞の転写因子媒介リプログラミングに続いて心筋細胞のサブタイプを評価します

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

2および3次元の胚様体中のマウス胚性幹細胞のレチノイン酸誘発神経分化の分析

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Dissection and Culture of Mouse Embryonic Kidney

マウス胎児腎臓の解剖および培養

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

傷害による老化と生体内での骨格筋におけるリプログラミングの評価

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

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 ...