Name: Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts (Video) | JoVE Journal
Uploaded: 2017-03-22T14:00:00.000+00:00
Duration: 9 min 29 s
Description: Learn direct reprogramming of mouse embryonic fibroblasts into induced cardiomyocyte subtypes using GHMT transcription factors. This method enables quantification of atrial, ventricular, and pacemaker cells for cardiac lineage studies.

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5 minutes

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 previously22,23, and place them into a Petri dish on ice with 1x phosphate-buffered saline (PBS) without Ca2+ or Mg2+.</li>
	</ol>
	</li>
	<li>Perform all subsequent steps in the tissue culture hood using sterile technique.
	<ol>
		<li>Remove the embryos from the uterus and amniotic sac using scissors and dissecting forceps. Keep the placenta attached for better handling. Pregnant CD-1 females typically give birth to between 10 and 14 pups.</li>
		<li>Using dissecting forceps, take the isolated embryos and quickly rinse them twice in 70% (v/v) EtOH. 
		NOTE: The washes should be fast to minimize cell death.</li>
		<li>Remove the head, limbs, tail, and internal organs, including the heart from the isolated embryos.</li>
		<li>Place the remaining tissue in a 10-cm dish with 1 mL of 1x PBS and finely mince using a sterile razor blade to approximately 1 mm3 in size.</li>
		<li>Transfer minced tissue into a 50 mL conical tube with PBS.</li>
		<li>Spin at 300 x g for 3 min. Carefully aspirate excess PBS.</li>
		<li>Add 1 mL of sterile 0.25% trypsin-EDTA per embryo. Incubate cells in a 37 &#176;C water bath for 15 min. Gently mix the tube every 4 min. Over-digestion of the tissue will significantly lower the yield.</li>
		<li>Vortex cell mixture at maximum speed (3,200 rpm) for 4 s.</li>
		<li>Add 2 mL of fibroblast media per embryo and mix. Filter through a 100 &#181;m cell strainer using a pipette to aid the cells through the strainer. Refer to Table 1 for the formulation of all subsequent mediums.</li>
		<li>Spin at 300 x g for 4 min. Carefully aspirate supernatant.</li>
		<li>Add 10 mL of fresh fibroblast media per 3 embryos and triturate 6-10 times.</li>
		<li>Plate the cells in 1 15-cm tissue culture dish for every 3 embryos prepared. Culture overnight in a 37 &#176;C, 5% CO2 incubator.</li>
		<li>After the overnight incubation, replace the media with fresh 30 mL of media per plate. Place cells back into the incubator overnight. 
		NOTE: Check for Hcn4-GFP+ cell contamination under a fluorescent microscope. The culture should be GFP- and only become GFP+ upon reprogramming.</li>
		<li>The next day, harvest cells with 3 mL of fresh pre-warmed 0.25% trypsin-EDTA. Count and freeze cells. Typically, freeze cells at 3 x 106 cells per mL. The expected yield should be 3 x 106 cells per embryo.</li>
	</ol>
	</li>
</ol>
2. Retrovirus Production and Reprogramming
Caution: The following protocol requires production and handling of infectious retroviruses. Perform the following steps in a Biosafety Level 2 cabinet under BSL-2 guidelines and sterile technique. Use 10% bleach to dispose of all materials exposed to retroviruses.
<ol>
	<li>Retrovirus production and MEFs preparation 
	NOTE: The following protocol is for retroviral production in 6-well plates and MEF infection in 24-well plates. For other formats, refer to Table 2. MEFs are plated at Day -1, so the timing will need to be coordinated appropriately for each experiment (Refer to section 2.3 and Figure 2).
	<ol>
		<li>Maintain Plat-E (PE) cells as per manufacturer&#39;s recommendations. Briefly, culture PE cells in DMEM supplemented with 10% FBS, 1 &#181;g/mL puromycin, 10 &#181;g/mL blasticidin, penicillin, and streptomycin. Passage cells 1:4 every two days when the culture reaches 70-90% confluency.</li>
		<li>Day -2: The day before transfection, seed Plat-E cells at 1 x 106 cells/well on a 6-well plate in transfection media. Cells should be 70-80% confluent at the time of transfection.</li>
	</ol>
	</li>
	<li>Transfection using a commercial transfection agent. 
	NOTE: The commercial reagents should be at room temperature (RT) before transfection. For the DNA transfection, add each retroviral plasmid DNA individually (G, H, M, and T) to form a GHMT cocktail9.
	<ol>
		<li>Day -1: In a 15 mL conical polystyrene tube, mix 60 &#181;L of reduced serum media with 6 &#181;L of transfection reagent per reaction for a 6-well plate format. Incubate the mixture for 5 min at room temperature. 
		NOTE: Since the transfection reagent used here binds to plastics, add directly to the reduced serum media to avoid any decrease in transfection efficiency.</li>
		<li>Add a total of 2 &#181;g of GHMT cocktail per reaction and gently tap to mix it. Do not vortex. Incubate the reaction for 15 min at RT.</li>
		<li>Add the mixture from step 2.2.3 to the PE cells in a drop wise manner.</li>
		<li>Incubate the transfected Plat-E cells overnight in a 37 &#176;C, 5% CO2 incubator. Record the time of transfection.</li>
	</ol>
	</li>
	<li>Seeding of Hcn4-GFP mouse embryonic fibroblasts.
	<ol>
		<li>1 h before plating MEFs, prepare a 24-well plate for immunocytochemistry.
		<ol>
			<li>Add a 12 mm fibronectin coverslip per well.</li>
			<li>Coat wells with 300 &#181;L of bovine collagen solution (e.g., SureCoat) and incubate in a 37 &#176;C incubator for 1 h.</li>
			<li>Aspirate coating solution immediately before MEF plating.</li>
		</ol>
		</li>
		<li>Thaw a frozen vial of Hcn4-GFP MEFs and wash x1 with pre-warmed fibroblast media at 500 x g for 5 min.</li>
		<li>Determine cell viability using trypan blue exclusion or similar dyes. Calculate the number of viable cells per mL of culture using the following formula: 
		% viable cells = [1.00 - (Number of blue cells/ Number of total cells)] x 100
		<ol>
			<li>Calculate total number of viable cells using the following formula: 
			Viable cells = % viable cells x dilution factor x 10,000 x total volume of cell suspension</li>
		</ol>
		</li>
		<li>Seed 3 x 104 cells per well onto a 24-well plate with previously prepared bovine collagen solution-fibronectin coverslip.</li>
	</ol>
	</li>
	<li>Transduction and reprogramming of MEFs 
	NOTE: According to the manufacturer&#39;s notes, properly maintained Plat-E cells produce an average titer of 1 x 107 infection units/mL. Although the titer is not directly measured for each experiment, a GFP control is always included as a surrogate for infection efficiency. High GFP expression and intensity (GFP+ &#62; 95%) typically correlates with successful GHMT-mediated generation of iCLMs.
	<ol>
		<li>Day 0: 24 h post-transfection, filter the PE retroviral medium through a 0.45 &#181;m-pore size surfactant-free cellulose acetate filter and transfer to a 15 mL conical tube. Add polybrene to a final concentration of 8 &#181;g/mL. Carefully replenish cells with 2 mL of fresh transfection medium. 
		NOTE: Cells easily detach from the plate if media is changed too rapidly.</li>
		<li>Aspirate the medium of the cultured MEFs and add the freshly collected retroviral medium; it should yield 1.7 mL of media per well of a 6-well plate. Add ~800 &#181;L per well of a 24-well plate. Return MEF plate to the incubator and incubate overnight.</li>
		<li>Day 1: Repeat steps 2.4.1 and 2.4.2. Discard cells after the 2nd virus collection. Return infected MEFs to the incubator and let them rest overnight.</li>
		<li>Day 2: 48 h post-induction, aspirate the cell conditioned media and wash x1 with 1x PBS. Add 500 &#181;L pre-warmed iCLM media per well of a 24 well plate.</li>
		<li>Replace iCLM media every 2 - 3 days. Process plate 14 days after viral induction for immunocytochemistry (ICC) analysis of cardiac reprogramming.</li>
	</ol>
	</li>
</ol>
3. Immunostaining of Reprogrammed MEFs
<ol>
	<li>14-days post-induction, carefully aspirate the media.</li>
	<li>Rinse each well with 300 &#181;L of ice-cold 1x PBS. Aspirate excess solution.</li>
	<li>Fix cells with 250 &#181;L of 4% paraformaldehyde (PFA) solution per well of a 24 well plate. Incubate 15 min at RT. 
	NOTE: Fixed cells can be stored in PBS at 4 &#176;C for 1 - 2 weeks before staining.</li>
	<li>Permeabilize cells by washing wells x3 with 300 &#181;L 0.1% PBS-TritonX 100 (PBST). Incubate 5 min at RT between washes. Aspirate excess solution after the last wash.</li>
	<li>Block for 10 min at RT with 1x Universal Block Buffer at 300 &#181;L/well.</li>
	<li>Prepare (ICC) staining buffer: Add 1:1 of 1x PBS and 1x Universal blocking buffer. Dilute primary antibodies in ICC staining buffer and incubate antibodies overnight at 4 &#176;C. Refer to the material section for recommended dilutions.
	<ol>
		<li>Stain one pair of slides with mouse &#945;-actinin, chicken &#945;GFP, and rabbit Nppa for iPM and iAM identification.</li>
		<li>Stain one pair of slides with mouse &#945;-actinin, chicken &#945;GFP, and rabbit Myl2 for iPM and iVM identification.</li>
	</ol>
	</li>
	<li>The following day, wash wells x3 with 300 &#181;L 0.1% PBST. Incubate 5 min at RT between washes. Aspirate excess solution after the last wash.</li>
	<li>Prepare the secondary antibody dilutions in ICC staining buffer. Refer to the material section for recommended dilutions. Incubate secondary antibodies 1 h at RT, protected from light.
	<ol>
		<li>Stain all slides with the following secondary antibodies: mouse Alexa-555, chicken Alexa-488, and rabbit Alexa-647.</li>
	</ol>
	</li>
	<li>Wash wells x3 with 300 &#181;L 0.1% PBST. Incubate 5 min at RT between washes. Protect from light.</li>
	<li>Add 2.4 &#181;L of mounting media containing 1.5 &#181;g/mL of 4&#39;,6-diamidino-2-phenylindole (DAPI) to a glass microscope slide. Carefully remove the coverslip from the well of the 24-well plate, remove excess solution, and transfer to the glass slide with mounting media. Gently press the coverslip to remove excess volume and air.</li>
	<li>Seal slides with preferred nail polish or plastic sealant. Store mounted slides at 4 &#176;C protected from light.</li>
</ol>
4. Identification of Cardiac Subtypes Using Confocal Microscopy
NOTE: For imaging, a confocal microscope equipped with at least 2 fluorescent detectors capable of spectral detection at 405, 488, 555, and 639 nm wavelengths is necessary in order to identify iPMs, iAMs, and iVMs. Image cells using a Plan-Apochromat 20X/0.75 objective or better. Using the manufacturer&#39;s image analysis software, scanning zoom images can achieve 40X-oil immersion quality images.
<ol>
	<li>Image library: take 8-bit images with DAPI, Alexa-488, Alexa-555, and Alexa-647 channels (ch.). Pixel dwell time of 6 s, 1024 frame size, line step at 2, and averaging of 2 is sufficient for high resolution images.</li>
	<li>For each slide, start from one edge and start scanning up and down in the red fluorescent channel (ch.) for &#945;-actinin+ Sarcomere+ cells (Refer to Figure 3 and Figure 5A for examples). Sarcomere striations are easier to identify visually in the 555 nm wavelength.
	<ol>
		<li>Once an &#945;-actinin+ Sarcomere+ cell has been identified, switch to the green ch. and keep note if it is positive (iPM). Switch to the computer to assess the far-red 647 ch. (iAM or iVM). 
		NOTE: Cells that are &#945;-actinin+/Sarcomere+/Hnc4-GFP+/Nppa-/Myl2- are designated as iPMs. GFP expression will be seen throughout the cell (Figure 4A).</li>
		<li>Stain slides with &#945;-actinin (mouse-Alexa555), Hcn4-GFP (chicken-Alexa488), Nppa (rabbit-Alexa647), and DAPI. Cells that are &#945;-actinin+/Sarcomere+/Hnc4-GFP-/Nppa+ are iAM. Nppa staining will appear perinuclear and punctate (Figure 4B).</li>
		<li>Stain slides with &#945;-actinin (mouse-Alexa555), Hcn4-GFP (chicken-Alexa488), Myl2 (rabbit-Alexa647). Cells positive for &#945;-actinin+/Sarcomere+/Hnc4-GFP-/Myl2+ are iVMs. Myl2 staining will exhibit a striated form along the sarcomere filament. Due to variations in the quality of the staining and the Z-plane, striations may not always be easily visible (Figure 4C).</li>
	</ol>
	</li>
</ol>
5. Quantification
NOTE: In order to assess the actual number of potentially reprogrammed MEFs, 2 wells of a 24-well plate are seeded in parallel to the experimental wells and are harvested one day after plating. The total number of cells plated is then determined by averaging the two wells. This becomes the actual total cells plated (aTotal).
<ol>
	<li>Sarcomere+ 
	<ol>
		<li>Visually inspect each cell on a coverslip for proper &#945;-actinin+/Sarcomere+ (Right panels Figure 3) and record (Figure 5B-i).</li>
		<li>Tabulate the total number of &#945;-actinin+/Sarcomere+ on each coverslip and divide by the actual total cells plated (aTotal) (Figure 5B-iii). For example, if aTotal = 12,500 cells, and 100 cells were &#945;-Actinin+/Sarcomere+ then, 0.8% of the plated MEFs were reprogrammed. An average reprograming experiment will yield 1% &#945;-Actinin+/Sarcomere+ cells (Figure 5C).</li>
	</ol>
	</li>
	<li>Subtype+ 
	NOTE: For the following steps, refer to Figure 5B/C for a representative iCLM quantification workflow. Briefly, for each sarcomere+ cell, tabulate if it is unique for either subtype (Figure 5B-i). Calculate % Subtype (Figure 5B-iii) by dividing the number of subtype+ cells over the average sarcomere+ cells x 100 (Figure 5B-i). To calculate the absolute % subtype efficiency (Figure 5B-iv), divide the subtype+ cell number from Figure 5B-i by the total number of cells seeded x 100 (Figure 5B-ii).
	<ol>
		<li>For each of the &#945;-actinin+/Sarcomere+ cells, assess if they are GFP+, Nppa+, or Myl2+.</li>
		<li>Tabulate total number of &#945;-actinin+/Sarcomere+/Hnc4-GFP+/Nppa-/Myl2-, actinin+/Sarcomere+/Hnc4-GFP-/Nppa+, and &#945;-actinin+/Sarcomere+/Hnc4-GFP-/Myl2+ cells (Figure 5B-i).</li>
		<li>To calculate the percentage of each subtype, divide the number of Subtype+ cells by the total number of &#945;-actinin+/Sarcomere+ cells in that well and multiply by 100. GHMT generates iPMs, iAMs, and iVMs at roughly equal ratios (Figure 5B-iii).</li>
		<li>To calculate the absolute number of subtype+ cells, divide the total number of subtype+ cells for the experimental condition by aTotal and multiply by 100 (Figure 5B-ii). On average iPMs represent 0.3% of the total infected cell population, iAMs 0.3%, and iVMs 0.25% (Figure 5B-iv).</li>
	</ol>
	</li>
</ol>

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<li>Yang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+conversion+of+mouse+and+human+fibroblasts+to+functional+melanocytes+by+defined+factors%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors.</a> Nat Commun. 5, 5807(2014).</li>
<li>Muraoka, N., Ieda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+reprogramming+of+fibroblasts+into+myocytes+to+reverse+fibrosis%5BTitle%5D)+AND+%22Annu+Rev+Physiol%22%5BJournal%5D)">Direct reprogramming of fibroblasts into myocytes to reverse fibrosis.</a> Annu Rev Physiol. 76, 21-37 (2014).</li>
<li>Coffin, J. M., Hughes, S. H., Varmus, H. E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCoffin%2C+JM+%22Retroviruses%22">Retroviruses</a>. Coffin, J. M., Hughes, S. H., Varmus, H. E. , (1997).</li>
<li>Bass, G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+image+analysis+identifies+signaling+pathways+regulating+distinct+signatures+of+cardiac+myocyte+hypertrophy%5BTitle%5D)+AND+%22J+Mol+Cell+Cardiol%22%5BJournal%5D)">Automated image analysis identifies signaling pathways regulating distinct signatures of cardiac myocyte hypertrophy.</a> J Mol Cell Cardiol. 52 (5), 923-930 (2012).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>DMEM</td><td>Sigma</td><td>D5796</td><td>Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td></tr><tr><td>Medium 199</td><td>Thermo Fisher Scientific</td><td>11150059</td><td>Component of iCLM media</td></tr><tr><td>Fetal bovine serrum (FBS)</td><td>Sigma</td><td>F2442</td><td>Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td></tr><tr><td>Insulin-Transferrin-Selenium G</td><td>Thermo Fisher Scientific</td><td>41400-045</td><td>Component of iCLM media</td></tr><tr><td>MEM vitamin solution</td><td>Thermo Fisher Scientific</td><td>11120-052</td><td>Component of iCLM media</td></tr><tr><td>MEM amino acids</td><td>Thermo Fisher Scientific</td><td>1601149</td><td>Component of iCLM media</td></tr><tr><td>Non-Essential amino acids</td><td>Thermo Fisher Scientific</td><td>11140-050</td><td>Component of iCLM media</td></tr><tr><td>Antibiotic-Antimycotics</td><td>Thermo Fisher Scientific</td><td>15240062</td><td>Component of iCLM media</td></tr><tr><td>B-27 supplement</td><td>Thermo Fisher Scientific</td><td>17504044</td><td>Component of iCLM media</td></tr><tr><td>Heat-Inactivated Horse Serum</td><td>Thermo Fisher Scientific</td><td>26050-088</td><td>Component of iCLM media</td></tr><tr><td>NaPyruvate</td><td>Thermo Fisher Scientific</td><td>11360-70</td><td>Component of iCLM media</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Fisher Scientific</td><td>1514022</td><td>Component of Plat-E media and fibroblast media</td></tr><tr><td>Puromycin&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A11139-03</td><td>Component of Plat-E media</td></tr><tr><td>Blasticidin&amp;nbsp;&amp;nbsp;</td><td>Gemini Bio-Products</td><td>400-128P</td><td>Component of Plat-E media</td></tr><tr><td>Glutamax</td><td>Thermo Fisher Scientific</td><td>35050-061</td><td>Component of Fibroblast media</td></tr><tr><td>Confocal laser scanning LSM700</td><td>Zeiss</td><td></td><td>For confocal analysis</td></tr><tr><td>FuGENE 6 transfection Reagent</td><td>Promega</td><td>E2692</td><td>Transfection reagent&amp;nbsp;</td></tr><tr><td>Opti-MEM Reduced Serum Medium</td><td>Thermo Fisher Scientific</td><td>31985-070</td><td>Transfection reagent&amp;nbsp;</td></tr><tr><td>Polybrene</td><td>Millipore</td><td>TR-1003-G</td><td>Induction reagent. Use at a final concentration of 8 &amp;mu;M/mL</td></tr><tr><td>Platinium-E (PE) Retroviral Packagin Cell Line, Ecotropic</td><td>CellBiolabs</td><td>RV-101</td><td>Retroviral pacaking cell line</td></tr><tr><td>Trypsin 0.25% EDTA</td><td>Thermo Fisher Scientific</td><td></td><td>For MEFs and Plat-E dissociation</td></tr><tr><td>Mouse anti &amp;alpha;-Actinin (Clone EA-53)</td><td>Sigma</td><td>A7811</td><td>Antibody for confocal analysis. Use at 1:200</td></tr><tr><td>Chicken anti-GFP IgY</td><td>Thermo Fisher Scientific</td><td>A10262</td><td>Antibody for confocal analysis. Use at 1:200</td></tr><tr><td>Rabbit Pab anti-NPPA&amp;nbsp;</td><td>Abgent</td><td>AP8534A</td><td>Antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Rabbit Pab anti Myl2 IgG&amp;nbsp;</td><td>ProteinTech</td><td>10906-1-AP</td><td>Antibody for confocal analysis. Use at 1:200</td></tr><tr><td>Vectashield solution with DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Vector Labs</td><td>H-1500</td><td>Dye for confocal analysis</td></tr><tr><td>Superfrost Plus Microscope slides</td><td>Thermo Fisher Scientific</td><td>12-550-15</td><td>25 x 75 x 1.0 mm</td></tr><tr><td>BioCoat Fibronectin 12 mm coverslips</td><td>NeuVitro Corp</td><td>GG-12-1.5</td><td>Coverslips for confocal analysis</td></tr><tr><td>100 &amp;mu;m cell strainer</td><td>Thermo Fisher Scientific</td><td>08-771-19</td><td></td></tr><tr><td>0.45 &amp;mu;m Syringes filters SFCA 25MM</td><td>Thermo Fisher Scientific</td><td>09-740-106</td><td>For virus filtration</td></tr><tr><td>6 mL Syringes</td><td>Covidien</td><td>8881516937</td><td>For virus filtration</td></tr><tr><td>Goat anti-Chicken IgY (H&amp;amp;L) A488</td><td>Abcam</td><td>AB150169</td><td>Secondary antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Donkey anti-rabbit A647 IgG(H+L)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A31573</td><td>Secondary antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Goat anti-mouse IgG(H+L) A555</td><td>Thermo Fisher Scientific</td><td>A21422</td><td>Secondary antibody for confocal analysis. Use at 1:400</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>93443-100ml</td><td>For cell permeabilization&amp;nbsp;</td></tr><tr><td>Dulbecco&#039;s PBS without CaCl&lt;sub&gt;2&lt;/sub&gt; and MgCl&lt;sub&gt;2&lt;/sub&gt; (D-PBS)</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Power Block 10x Universal Blocking reagent</td><td>Thermo Fisher Scientific</td><td>NC9495720</td><td>Dilute to 1x in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>16% Paraformaldehyde aqueous solution (PFA)</td><td>Electro Microscopy Sciences&amp;nbsp;</td><td>15710</td><td>Use at 4% diluted in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>6 cm&amp;nbsp; plates</td><td>Olympus</td><td>25-260</td><td></td></tr><tr><td>6-well plates</td><td>Genesee Scientific</td><td>25-105</td><td></td></tr><tr><td>24-well plates</td><td>Genesee Scientific</td><td>25-107</td><td></td></tr><tr><td>10 cm Tissue culture dishes</td><td>Corning</td><td>4239</td><td></td></tr><tr><td>15 cm&amp;nbsp; Tissue culture dishes</td><td>Thermo Fisher Scientific</td><td>5442</td><td></td></tr><tr><td>15 mL Conical tubes</td><td>Corning</td><td>4308</td><td></td></tr><tr><td>50 mL Conical tubes</td><td>Corning</td><td>4249</td><td></td></tr><tr><td>0.4% Trypan blue solution</td><td>Sigma</td><td>T8154</td><td>For viability</td></tr><tr><td>Ethyl Alcohol 200 proof</td><td>Thermo Fisher Scientific</td><td>7005</td><td></td></tr><tr><td>Bleach</td><td>Thermo Fisher Scientific</td><td>6009</td><td></td></tr></tbody></table>

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

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...

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 ...

The overall goal of this procedure is to generate and quantify a diverse set of induced cardiomyocyte subtypes. This method can help answer key, fundamental questions regarding subtype-specific lineage conversion during cardiac reprogramming such as the necessary cues that regulate sarcomere assembly and subtype specification. The main advantage of this technique is that it provides the method for quantified effects on cardio subtype specification which is required to perform mechanisis studies.To begin, isolate mouse embryonic fiberblasts from stage E 12.5 embryos. Store them in liquid nitrogen as described in the accompanying text protocol. Next, harvest plat-E cells and plate them at one million cells per well into a six well plate so that the cells reach 70 to 80%confluency in 24 hours.Cover the cells with transfaction media and incubate them under standard culture conditions. On experiment day minus one, take a 15 milliliter conical polystyrene tube and mix 60 microliters of reduced serum media with six microliters of transfection reagent per reaction. Incubate the mixture for five minutes at room temperature.Since the transfection reagent use your add your reagent directly to the reduced serum media to avoid any decrease in transfection efficiency. Next, add a total of two micrograms of GHMT cocktail per reaction, and gently tap the tube to mix it. Incubate the reaction for 15 minutes at room temperature and then add the mixture to the plat-E cells in the drop wise manner.Record the date and time of transfection. Incubate the transfected plat-E cells overnight. One hour before plating the mouse embryonic fibroblast, prepare a 24 well plate for imunno-cytochemistry by first adding 12 millimeter fibronectin coated cover slips to each well.Next, add 300 microliters of bovine collagen solution to each well and incubate the plate for one hour. Near the end of the incubation, thaw a frozen vial of HCN4GFP mouse embryonic fibroblast. Quick thaw the cells, wash them once with pre-warmed fiberblast media and count the concentration of live cells.Then aspirate the coating solution in the 24 well plate and immediately plate 30, 000 cells per well onto the collagen and fibronectin coated cover slips. 24 hours post-transfection, filter the retro-viral medium through a 0.45 micron pore size surfectant free cellulose acetate filter, and transfer it to a 15 milliliter conical tube. Then, add polybrene to a final concentration of eight micrograms per milliliter.Carefully replenish the plat-E cells with two milliliters of fresh transfection medium. Changing the media too rapidly may cause the cells to detatch. Properly maintained plat-E cells produce an average of ten million infection units per milliliter.High GFP expression and intensity in more than 95%of the cells, typically correlates with a successful GHNT mediated generation of induced cardiomyocyte-like myocytes. Next, aspirate the medium of the cultured embryonic fibroblasts and add the freshly collected retroviral medium. Then return meth plate to the incubator and incubate overnight.The next day, again transfer the media from the plat-E cells to the embryonic fibroblasts. Discard the plat-E cells after the second virus collection. 48 hours post induction, aspirate the cell condition media and wash the cells once with PBS.Then add 500 microliters of pre-warmed induced cardio myocyte-like myocyite media per well of the 24 well plate. Replace the media every two to three days for the next 12 days. 14 days post induction, carefully aspirate the media and rinse each well with 300 microliters of ice cold PBS.Then removed the PBS and add 250 microliters of a 4%paraformaldehyde solution per well and incubate the cells for 15 minutes at room temperature. Next, permeabalize the cells by washing the wells three times for five minutes with 300 microliters of 0.1%tritonex 100 and PBS. Aspirate the excess permiableization solution after the last wash, and then add a universal blocking buffer at 300 microliters per well.Block for 10 minutes at room temperature. Stain one pair of slides overnight with mouse anti-alpha actenin, chicken anti-GFP and rabbit anti-NPPA antibodies to identify induced pacemaker-like myocytes in induced atrial-like myocytes. The following day, wash the wells and add the appropriate secondary antibody as described in the accompanying text protocol.Following additional washes, add 2.4 microliters of mounting media to a glass microscope slide. Take a cover slip from the 24 well plate and remove the excess solution. Then transfer it to the glass slide with the mounting media.For each slide, start from one edge and start scanning up and down in the red fluorescent channel to identify alpha-actinin positive sarcomere positive cells. Sarcomere striations are easiest to identify in the 555 nanometer wavelength. Once identified, switch to the green channel and keep note if the cells are positive.Then switch to the computer to asses the far red 647 nanometer wavelength to look for NPPA or MYL2 staining. Cells that are alpha-actinin positive, sarcomere positive, Hnc4-GFP negative and Nppa positive are induced atrial-like myocytes. Nppa staining will appear paranuclear and punctate.First, count the number of cells 24 hours after seeding as described in the accompanying text protocol. Next, visually inspect each cell on a cover slip for proper alpha-actinin and sacromere staining and record the number of cells positively stained for both markers. Then, divide by the actual total cells plated.An average reprogramming experiment will yield approximately one percent of cells that are both alpha-actinin and sarcomere positive. By day 14, the experiment can be stopped and assessed for sarcomere organization. This is identified by the positive alpha-actinin staining shown here in red.Only cells that exhibit sarcomere organization are capable of spontaneous beating, so this is a good first check. Shown here are induced pacemaker-like cardiomyocites from Hcn4-GFP reporter mice. Three cells from this view exhibit sarcomere organization.Three cell clusters positive for Hcn4-GFP are present in the center of the image and can be idenfied as a pacemaker cell type. Once cell at the bottom left can be observed with alpha-actinin staining but no sarcomere organization or identifiable subtype marker. In this population of induced ventricular like cardiomyocytes, the central cell exhibits sarcomere organization, this cell also stains positive for the myocin light chain marker MYL-2 which is a marker for ventricular cardio myocytes.Once mastered, the plating and the transfection of the plat-E cells can be performed in one hour. The induction of the mouse embryonic fibroblasts in one to two hours and the myto quantification of a full 24 well plate in about eight hours. While attempting this procedure, it's important to remember that healthy mouse embryonic fibroblasts and high titer viruses are key for a successful reprogramming experiment.After watching this video, you should have a good understanding of how to generate and quantify individual induced cardiomyocyte subtypes.

assessing-cardiomyocyte-subtypes-following-transcription-factor

Research

JoVE Journal

Developmental Biology

Single cell expression profiling allows the detailed gene expression analysis of individual cells.  We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

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.

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

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

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.

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

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-β signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

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

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

Learn direct reprogramming of mouse embryonic fibroblasts into induced cardiomyocyte subtypes using GHMT transcription factors. This method enables quantification of atrial, ventricular, and pacemaker cells for cardiac lineage studies.

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

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