Name: assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts
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Description: Watch this Scientific Journal Video about Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts at JoVE.com

A.F.-P. was supported by the National Science Foundation Graduate Research Fellowship under Grant No.2015165336. N.V.M was supported by grants from the NIH (HL094699), Burroughs Wellcome Fund (1009838), and the March of Dimes (#5-FY14-203). We acknowledge Young-Jae Nam, Christina Lubczyk, and Minoti Bhakta for their important contributions to protocol development and data analysis. We also thank John Shelton for valuable technical input and members of the Munshi lab for scientific discussion.

動物プラクティスを含む全ての実験手順は、UTサウスウェスタン医療センターの施設内動物管理使用委員会によって承認されました。 HCN4-GFP E12.5マウス胚線維芽細胞（MEFの）の1の単離<ol><li>ホモ接合HCN4-GFPの雄とCD-1雌との間の時限交配を設定します。 </li><li>二酸化炭素安楽死とその後の頸椎脱臼によりE12.5で妊娠中の女性を生け贄に捧げます。 <ol><li> 2+、</s...

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本研究は、心臓転写のレトロウイルス媒介発現を介して、心臓のサブタイプの多様なセットにしたMEFの変換のためのダイレクトリプログラミング戦略を提供することはGATA4、MEF2C、TBX5、およびHand2（GHMT）要因。 PM固有レポーターマウスと組み合わせて多重免疫染色法を用いて、我々は、単一細胞の解像度でIAM、IVMS、およびIPMSを識別することができます。このようなアッセイは、サブタイプの...

心臓は胚1、2に開発した最初の機能的器官です。循環器系に関連して、開発中に、酸素、栄養素、および廃棄物処理機構に供給する。三週間受精後、人間の心は初めて打つとその適切な規制は、心筋細胞（のCM）によって維持されています。これらの特殊化した細胞の不可逆的損失は、したがって、進行性の心不全の根底にある基本的な問題です。このようなゼブラフィッシュおよびアフリカツメガエルのようないくつかの生物は、心臓の再生の可能性を有しているが、大人の哺乳類の心臓は3、5、6、より限定されています。このように、心の重要な機能を考えると、心臓病は米国だけで60万人が死亡7を占め、世界における死亡の主な原因であることは驚くべきではありません。ザrefore、効率的に負傷した心筋を修理または交換するために、細胞ベースの治療は臨床上非常に興味深いものです。 山中ら8の精液の研究は、4つの転写因子の強制発現は、多能性幹細胞に完全に分化した線維芽細胞に変換するのに十分であることを示しました。しかし、すべての多能性幹細胞戦略の腫瘍形成能力は、治療目的のためのそれらの使用における重大な関心事となっています。これは、多能性段階を回避しながら細胞を分化転換するための代替方法を検索するための科学的なフィールドをやる気。最近、いくつかのグループは、転写の異所性発現は、GATA4、MEF2C、TBX5、および後に、Hand2（GMTとGHMT因子を用いて誘導心筋細胞様細胞（iCLMs）にマウス線維芽細胞の直接変換を表示することにより、この戦略の実現可能性を示していますそれぞれ）9,10。 Furthermore、同じ戦略は、 インビボおよびヒト由来の組織9、11、12で行うことができます。最近の研究では、追加の因子またはさらなる心臓再プログラミング効率13、14、15を改善するために調節することができるシグナル伝達経路を強調しています。まとめると、これらの研究は、再生治療のために向かう分化転換の可能性を示します。しかし、方法論的差異16にCMの再プログラミング、未知の分子機構、一貫性のない再現性の低効率、およびiCLMsの不均一な性質は、アドレス指定されないままです。 直接iCLM不均一性を評価するために、我々は、サルコメアの開発および心臓系統specificatioの同定のために離散的で堅牢な単一細胞アッセイを設計しましたn-2の機能的心筋細胞の必要な特性。それらの場所でユニークな電気的特性によって定義されたCMの少なくとも三つの主要なタイプは、心臓である：心房（AM）は、心室（VM）及びペースメーカー（PM）17、18、19、20。オーケストレーションの組み合わせで、それらは、血液の適切なポンピングを可能にします。心臓損傷の間、一つまたはすべてのサブタイプが影響を受ける可能性があり、および細胞治療のタイプは、ケースバイケースで対処する必要があります。少し作業は、サブタイプの仕様を調節する分子メカニズムを研究するために行われている間、現在、ほとんどの戦略は、心筋細胞の全体的な世代に焦点を当てています。 以下の研究は、適切によく組織サルコメアを定量化し、心筋細胞のサブタイプの多様なセットを識別する方法について詳しく説明します。ペースメーカー（PM）特異的レポーターマウスを用いて、Iを適用することができます誘導された心房のような筋細胞（IAM）、誘導された心室のような筋細胞（IVM）、および誘導されたPM-様筋細胞（IPMS）21を区別するためにmmunocytochemicalアプローチ。我々の観察に基づいて、サルコメア組織を示す細胞のみが自発的な拍動することが可能です。このユニークな再プログラミングプラットフォームは役割を評価することができます サルコメア組織、サブタイプの仕様、および単一セルの解像度でCMの再プログラミングの効率の特定のパラメータの。

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">DMEM</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">D5796</td>
			<td style="width:347px;">Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Medium 199</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11150059</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Fetal bovine serrum (FBS)</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">F2442</td>
			<td style="width:347px;">Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Insulin-Transferrin-Selenium G</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">41400-045</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">MEM vitamin solution</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11120-052</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">MEM amino acids</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">1601149</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Non-Essential amino acids</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11140-050</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Antibiotic-Antimycotics</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15240062</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">B-27 supplement</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17504044</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Heat-Inactivated Horse Serum</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">26050-088</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">NaPyruvate</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11360-70</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Penicillin/Streptomycin</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">1514022</td>
			<td style="width:347px;">Component of Plat-E media and fibroblast media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Puromycin&#160;</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A11139-03</td>
			<td>Component of Plat-E media</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Blasticidin&#160;&#160;</td>
			<td style="width:91px;">Gemini Bio-Products</td>
			<td>400-128P</td>
			<td>Component of Plat-E media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Glutamax</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>35050-061</td>
			<td>Component of Fibroblast media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Confocal laser scanning LSM700</td>
			<td style="width:91px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>For confocal analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">FuGENE 6 transfection Reagent</td>
			<td style="width:91px;">Promega</td>
			<td style="width:119px;">E2692</td>
			<td>Transfection reagent&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Opti-MEM Reduced Serum Medium</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">31985-070</td>
			<td>Transfection reagent&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Polybrene</td>
			<td style="width:91px;">Millipore</td>
			<td style="width:119px;">TR-1003-G</td>
			<td>Induction reagent. Use at a final concentration of 8um/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Platinium-E (PE) Retroviral Packagin Cell Line, Ecotropic</td>
			<td style="width:91px;">CellBiolabs</td>
			<td style="width:119px;">RV-101</td>
			<td>Retroviral pacaking cell line</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Trypsin 0.25% EDTA</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td>For MEFs and Plat-E dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti &#945;-Actinin (Clone EA-53)</td>
			<td style="width:91px;">Sigma</td>
			<td>A7811</td>
			<td>Antibody for confocal analysis. Use at 1:200</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Chicken anti-GFP IgY</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A10262</td>
			<td>Antibody for confocal analysis. Use at 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit Pab anti-NPPA&#160;</td>
			<td style="width:91px;">Abgent</td>
			<td>AP8534A</td>
			<td>Antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit Pab anti Myl2 IgG&#160;</td>
			<td style="width:91px;">ProteinTech</td>
			<td>10906-1-AP</td>
			<td>Antibody for confocal analysis. Use at 1:200</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Vectashield solution with DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:91px;">Vector Labs</td>
			<td style="width:119px;">H-1500</td>
			<td>Dye for confocal analysis</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Superfrost Plus Microscope slides</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12-550-15</td>
			<td>25 x 75 x 1.0 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">BioCoat Fibronectin 12mm coverslips</td>
			<td style="width:91px;">NeuVitro Corp</td>
			<td>GG-12-1.5</td>
			<td>Coverslips for confocal analysis</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">100um cell strainer</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">0.45um Syringes filters SFCA 25MM</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">09-740-106</td>
			<td>For virus filtration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">6ml Syringes</td>
			<td style="width:91px;">Covidien</td>
			<td>8881516937</td>
			<td>For virus filtration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-Chicken IgY (H&#38;L) A488</td>
			<td style="width:91px;">Abcam</td>
			<td>AB150169</td>
			<td style="width:347px;">Secondary antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Donkey anti-rabbit A647 IgG(H+L)&#160;</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A31573</td>
			<td style="width:347px;">Secondary antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Goat anti-mouse IgG(H+L) A555</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A21422</td>
			<td style="width:347px;">Secondary antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Triton X-100</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">93443-100ml</td>
			<td>For cell permeabilization&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Dulbecco&#39;s PBS without CaCl2 and MgCl2 (D-PBS)</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Power Block 10X Universal Blocking reagent</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">NC9495720</td>
			<td>Dilute to 1X in H20</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:319px;">16% Paraformaldehyde aqueous solution (PFA)</td>
			<td style="width:91px;">Electro Microscopy Sciences&#160;</td>
			<td style="width:119px;">15710</td>
			<td>Use at 4% diluted in dH20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">6 cm&#160; plates</td>
			<td style="width:91px;">Olympus</td>
			<td style="width:119px;">25-260</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">6-well plates</td>
			<td style="width:91px;">Genesee Scientific</td>
			<td style="width:119px;">25-105</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">24-well plates</td>
			<td style="width:91px;">Genesee Scientific</td>
			<td style="width:119px;">25-107</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">10 cm Tissue culture dishes</td>
			<td style="width:91px;">Corning</td>
			<td>4239</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">15 cm&#160; Tissue culture dishes</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>5442</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">15 ml Conical tubes</td>
			<td style="width:91px;">Corning</td>
			<td style="width:119px;">4308</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">50 ml Conical tubes</td>
			<td style="width:91px;">Corning</td>
			<td style="width:119px;">4249</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">0.4% Trypan blue solution</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">T8154</td>
			<td>For viability</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Ethyl Alcohol 200 proof</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">7005</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Bleach</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">6009</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

PM固有のレポーターマウスを利用して、我々は、 図1に示されるように、多様な内因性の筋細胞を同定するための多重免疫戦略を開発しました。 図2に示す書き換え手順に続いて、サブタイプ特異的のCMの誘導は、低レートではあるが、早ければ4日目21として検出することができます。 14日目では、実験は、サルコメア組織（...

Watch this Scientific Journal Video about Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts at JoVE.com

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

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

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 ...

Research

JoVE Journal

Developmental Biology

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

エレクトロポレーションによりマウス胎児蝸牛外植の文化と遺伝子導入

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

胚性マウスの心臓に免疫蛍光を用いて、心筋細胞の開発の分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

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 ...

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

心筋細胞のDNA合成と倍数性の同時評価：心筋細胞再生の定量化を支援する方法および売上高

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

体細胞の再プログラミングの反応速度測定とリアルタイムの可視化

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

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 ...

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 ...

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

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 ...

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 ...