We are grateful for expert technical assistance from the UNC Flow Cytometry Core and UNC Microscopy Core. We thank members of the Qian lab and the Liu lab for helpful discussions and critical reviews of the manuscript. This study was supported by NIH/NHLBI R00 HL109079 grant to Dr. Liu and American Heart Association (AHA) Scientist Development Grant 13SDG17060010 and the Ellison Medical Foundation (EMF) New Scholar Grant AG-NS-1064-13 to Dr. Qian.

The protocol outlined here uses neonatal mice. Animal care and experiments are performed in accordance with the guidelines established by The Division of Laboratory Animal Medicine (DLAM) at University of North Carolina, Chapel Hill.
1. Preparation of Buffers and Media
<ol>
	<li>Prepare fibroblast (FB) medium: Supplement 500 ml IMDM media with 100 ml Fetal Bovine Serum (FBS) and 6 ml penicillin/streptomycin.</li>
	<li>Prepare iCM medi...

<ol>
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The authors have nothing to disclose.

For successful iCM generation when using this protocol, there are several important factors that have an impact on the overall efficiency. Particularly the conditions of starting fibroblasts and the quality of retrovirus encoding MGT can greatly affect the reprogramming efficiency.
It is important to generate fibroblasts as fresh and healthy as possible. For explant culture method, fibroblasts can be used before seven days after the explants were plated on dishes. For enzyme digestion method, ...

Cardiovascular disease remains the leading cause of death worldwide, accounting for 17.3 million deaths per year1. Loss of cardiomyocytes resulting from myocardial infarction (MI) or progressive heart failure is a major cause of morbidity and mortality2. Due to limited regenerative capacity, adult mammalian hearts usually suffer from impaired pump function and heart failure following injury3-6. As such, efficient (re)generation of cardiomyocytes in vivo and in vitro for treatment of heart disease and for disease modeling is a critical issue needing to be addressed.
Recent development of direct reprogramming, which directly reprograms cells from one differentiated phenotype to another without transitioning through the pluripotent state, offers a promising alternative approach for regenerative medicine. The mammalian heart contains abundant cardiac fibroblasts (CFs), which account for approximately half of the cells in heart and massively proliferate upon injury7-9. Thus, the vast pool of CFs could serve as an endogenous source of new CMs for regenerative therapy if they could be directly reprogrammed into functional CMs. It has been shown that a combination of transcription factors, such as Gata4 (G), Mef2c (M) and Tbx5 (T), with or without microRNAs or small molecules can reprogram fibroblasts into iCMs10-26. Importantly, this conversion can also be induced in vivo, and results in an improvement in cardiac function and a reduction in scar size in an infarcted heart16,27-29. These studies indicate that direct cardiac reprogramming may be a potential avenue to heal an injured heart. However, the low efficiency of iCM reprogramming has become a major hurdle for further mechanistic studies. In addition, the reproducibility of cardiac reprogramming is another controversial issue of this technology11,30,31. 
Very recently, we generated a complete set of polycistronic constructs encoding G,M,T in all possible splicing orders with identical 2A sequences in a single mRNA. These polycistronic constructs yielded varied G, M and T protein expression levels, which led to significantly different reprogramming efficiency25. The most efficient construct, named MGT, which showed a relatively high Mef2c and low Gata4 and Tbx5 expression, significantly improved reprogramming efficiency and produced large amounts of iCMs with CM markers expression, robust calcium oscillation and spontaneous beating25. Moreover, by using MGT polycistronic construct, our study avoided the use of multiple vectors and generated cells with homogenous expression ratio of G,M,T, thus providing an improved platform for cardiac reprogramming research. To increase experimental reproducibility, here we describe in detail how to isolate fibroblasts, produce retrovirus carrying MGT cassette, generate iCMs and evaluate the reprogramming efficiency.

<table border="0" cellpadding="0" cellspacing="0" width="1088">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">anti-cardiac troponin T</td>
			<td style="width:236px;">Thermo Scientific</td>
			<td style="width:200px;">MS-295-PO</td>
			<td style="width:191px;">1:200 for FACS and 1:400 for ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-GFP</td>
			<td>Life Technologies</td>
			<td style="width:200px;">A11122</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti- aActinin</td>
			<td>Sigma-Aldrich</td>
			<td>A7811</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Connexin43</td>
			<td>Sigma-Aldrich</td>
			<td>C6219</td>
			<td>1:500 for&#160; ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anit-Mef2c</td>
			<td>Abcam</td>
			<td style="width:200px;">ab64644</td>
			<td>1:1000 for ICC&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Gata4</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:200px;">sc-1237</td>
			<td>1:200 for ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-Tbx5</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-17866</td>
			<td>1:200 for ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor 488&#8211;conjugated donkey anti-rabbit IgG</td>
			<td>Jackson ImmunoResearch Inc</td>
			<td>711-545-152</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alexa Fluor 647&#8211;conjugated donkey anti-mouse IgG</td>
			<td>Jackson ImmunoResearch Inc</td>
			<td>715-605-150</td>
			<td>1:500 for both FACS and ICC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">Cytofix/Cytoperm kit for intracellular staining</td>
			<td style="width:236px;">BD Biosciences</td>
			<td style="width:200px;">554722</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rhod-3 Calcium Imaging Kit</td>
			<td>Life Technologies</td>
			<td style="width:200px;">R10145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thy1.2 microbeads</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-049-101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vectashield solution with DAPI</td>
			<td>Vector labs</td>
			<td style="width:200px;">H-1500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td style="width:200px;">F-2442</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.05%)</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">25-052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PRMI1640 medium</td>
			<td>Life Technologies</td>
			<td style="width:200px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">B27 supplement</td>
			<td>Life Technologies</td>
			<td style="width:200px;">17504-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IMDM</td>
			<td>Life Technologies</td>
			<td style="width:200px;">12440-053</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM Reduced Serum Medium</td>
			<td>Life Technologies</td>
			<td style="width:200px;">31985-070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199 medium</td>
			<td>Life Technologies</td>
			<td style="width:200px;">10-060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM, high glucose</td>
			<td>Life Technologies</td>
			<td style="width:200px;">10-013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-streptomycin</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">30-002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Non-essential amino acids</td>
			<td>Life Technologies</td>
			<td style="width:200px;">11130-050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td style="width:200px;">11668500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">blasticidin</td>
			<td>Life Technologies</td>
			<td style="width:200px;">A11139-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">puromycin</td>
			<td>Life Technologies</td>
			<td style="width:200px;">A11138-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase II</td>
			<td>Worthington</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">polybrene</td>
			<td>Millipore</td>
			<td style="width:200px;">TR-1003-G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td style="width:236px;">Fisher</td>
			<td style="width:200px;">BP151-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CaCl2</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>PX1405</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Na2HPO4</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>S7907</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td>G6152</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin</td>
			<td>Fisher</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">paraformaldehyde</td>
			<td>EMS</td>
			<td style="width:200px;">15714</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Retrovirus Precipitation Solution</td>
			<td>ALSTEM</td>
			<td style="width:200px;">VC-200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.4%Trypan blue solution</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:200px;">T8154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">gelatin</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:200px;">G1393</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s PBS without CaCl2 and MgCl2 (D-PBS, 1x)</td>
			<td style="width:236px;">Sigma-Aldrich</td>
			<td style="width:200px;">D8537</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS (Hanks Balanced Salt Solution)</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">21022</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LS column</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 &#956;m cellulose acetate filter&#160;</td>
			<td>Thermo Scientific</td>
			<td style="width:200px;">190-2545</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24-well plates</td>
			<td style="width:236px;">Corning</td>
			<td style="width:200px;">3524</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10cm Tissue culture dishes&#160;</td>
			<td>Thermo Scientific</td>
			<td style="width:200px;">172958</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60mm center well culture dish&#160;&#160;</td>
			<td>Corning</td>
			<td style="width:200px;">3260</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 Well Clear V-Bottom 2mL Polypropylene Deep Well Plate</td>
			<td>Denville Scientific</td>
			<td style="width:200px;">P9639</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">Polystyrene round-bottom tubes with cell-strainer cap&#160;</td>
			<td style="width:236px;">BD Biosciences</td>
			<td style="width:200px;">352235</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td style="width:236px;">Eppendorf</td>
			<td style="width:200px;">5810R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortexer MINI</td>
			<td style="width:236px;">VWR</td>
			<td style="width:200px;">58816-121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EVOS&#174; FL Auto Cell Imaging System</td>
			<td>Life Technologies</td>
			<td style="width:200px;">AMAFD1000</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-042-303</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MidiMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td style="width:200px;">130-042-302</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:461px;">Round glass cover slip</td>
			<td style="width:236px;">Electron Microscopy Sciences</td>
			<td style="width:200px;">72195-15</td>
			<td></td>
		</tr>
	</tbody>
</table>

improved generation of induced cardiomyocytes using a polycistronic construct expressing optimal ratio of gata4, mef2c and tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

The reprogramming steps are summarized by schematic in Figure 1. After MGT transduction, GFP expression in reprogramming cells could be detected as early as day 3. Puromycin selection of transduced cells starts from day 3 and is maintained during the first two weeks if pMx-puro-MGT construct is used. By day 10 to day 14, expression of cardiac markers like cTnT and &#945;Actinin could be detected by both ICC (Figure 2B, step 5) and FACS (Figure 2A, step 6), indicating tha...

Watch this Scientific Journal Video about Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5 at JoVE.com

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

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

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

improved-generation-induced-cardiomyocytes-using-polycistronic

Research

JoVE Journal

Developmental Biology

8.6K visualizações.  University of North Carolina, Chapel Hill. We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming. 