Name: assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts
Uploaded: 2017-03-22T14:00:00
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.

All experimental procedures involving animal practices were approved by the Institutional Animal Care and Use Committee at UT Southwestern Medical Center.
1. Isolation of Hcn4-GFP E12.5 Mouse Embryonic Fibroblast (MEFs)
<ol>
	<li>Set up timed matings between homozygous Hcn4-GFP males and CD-1 females.</li>
	<li>Sacrifice pregnant female at E12.5 by carbon dioxide euthanasia and subsequent cervical dislocation.
	<ol>
		<li>Remove uterine horns with dissecting forceps as described previously<s...

<ol>
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The present study provides a direct-reprogramming strategy for conversion of MEFs into a diverse set of cardiac subtypes via retrovirus-mediated expression of the cardiac transcription factors Gata4, Mef2c, Tbx5, and Hand2 (GHMT). Using a multiplex immunostaining approach in combination with a PM-specific reporter mouse, we are able to identify iAM, iVMs, and iPMs at single cell resolution. Such an assay allows for an experimental in vitro system capable of isolating the contributions of individual transcription...

The heart is the first functional organ to develop in the embryo1,2. In conjunction with the circulatory system, it supplies oxygen, nutrients, and a waste disposal mechanism during development. Three weeks after fertilization, the human heart beats for the first time and its proper regulation is maintained by cardiomyocytes (CMs). The irreversible loss of these specialized cells is therefore the fundamental issue underlying progressive heart failure. While some organisms such as the zebrafish and Xenopus have the potential for cardiac regeneration, the adult mammalian heart is more limited3,5,6. Thus, given the critical function of the heart, it is not astonishing that heart disease is the leading cause of death in the world, accounting for 600,000 deaths in the United States alone7. Therefore, cell-based therapies to efficiently repair or replace the injured myocardium are of great clinical interest.
The seminal study of Yamanaka and colleagues8 showed that forced expression of four transcription factors is sufficient to convert fully differentiated fibroblast cells to pluripotent stem cells. However, the tumorigenic capacity of all pluripotent stem cell strategies has been a critical concern in their use for therapeutic purposes. This motivated the scientific field to search for alternative methods to transdifferentiate cells while avoiding a pluripotent stage. Recently, several groups have shown the feasibility of this strategy by displaying direct conversion of mouse fibroblasts to induced cardiomyocyte-like cells (iCLMs) with the ectopic expression of the transcription factors Gata4, Mef2c, Tbx5, and later on, Hand2 (GMT and GHMT, respectively)9,10. Furthermore, the same strategy can be performed in vivo and in human-derived tissues9,11,12. Recent studies have highlighted additional factors or signaling pathways that can be modulated to further improve cardiac reprogramming efficiency13,14,15. Taken together, these studies demonstrate the potential of directed transdifferentiation for regenerative therapies. However, the low efficiency of CM reprogramming, the unknown molecular mechanisms, inconsistent reproducibility due to methodological differences16, and the heterogeneous nature of iCLMs remain unaddressed.
In order to directly evaluate iCLM heterogeneity, we designed a discrete and robust single-cell assay for the identification of sarcomere development and cardiac lineage specification-two necessary characteristics of functional cardiomyocytes. There are at least three major types of CM in the heart as defined by their location and unique electrical properties: atrial (AM), ventricular (VM) and pacemaker (PM)17,18,19,20. In an orchestrated combination, they allow the proper pumping of blood. During heart injury, one or all subtypes might be affected, and the type of cell therapy would need to be addressed on a case-by-case basis. Currently, most strategies focus on the overall generation of cardiomyocytes, while little work is being done to study the molecular mechanisms that regulates subtype specification.
The following study details how to properly quantify well-organized sarcomeres and identify a diverse set of cardiomyocyte subtypes. Using a pacemaker (PM)-specific reporter mouse, we are able to apply an immunocytochemical approach to distinguish induced atrial-like myocytes (iAM), induced ventricular-like myocytes (iVM), and induced PM-like myocytes (iPMs)21. Based on our observations, only cells that exhibit sarcomere organization are capable of spontaneous beating. This unique reprogramming platform allows for assessing the role of certain parameters in sarcomere organization, subtype specification, and efficiency of CM reprogramming at single-cell resolution.

<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.

Taking advantage of the PM-specific reporter mouse, we developed a multiplex immunostaining strategy to identify diverse endogenous myocytes as depicted in Figure 1. Following the reprogramming steps shown in Figure 2, induction of subtype-specific CMs can be detected as early as day 421, albeit at a low-rate. By day 14, the experiment can be stopped and assessed for sarcomere organization (Figure 3) and subtype-sp...

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.

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

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

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

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

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

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

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

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

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

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

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

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

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