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>

<ol><li>Sissman, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developmental+landmarks+in+cardiac+morphogenesis%3A+comparative+chronology%5BTitle%5D)+AND+%22Am+J+Cardiol%22%5BJournal%5D)">Developmental landmarks in cardiac morphogenesis: comparative chronology.</a> Am J Cardiol. 25 (2), 141-148 (1970).</li>
<li>Buckingham, M., Meilhac, S., Zaffran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Building+the+mammalian+heart+from+two+sources+of+myocardial+cells%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Building the mammalian heart from two sources of myocardial cells.</a> Nat Rev Genet. 6 (11), 826-835 (2005).</li>
<li>Ali, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Existing+cardiomyocytes+generate+cardiomyocytes+at+a+low+rate+after+birth+in+mice%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Existing cardiomyocytes generate cardiomyocytes at a low rate after birth in mice.</a> Proc Natl Acad Sci U S A. 111 (24), 8850-8855 (2014).</li>
<li>Bergmann, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidence+for+cardiomyocyte+renewal+in+humans%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Evidence for cardiomyocyte renewal in humans.</a> Science. 324 (5923), 98-102 (2009).</li>
<li>Senyo, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mammalian+heart+renewal+by+pre-existing+cardiomyocytes%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Mammalian heart renewal by pre-existing cardiomyocytes.</a> Nature. 493 (7432), 433-436 (2013).</li>
<li>Lin, Z., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strategies+for+cardiac+regeneration+and+repair%5BTitle%5D)+AND+%22Sci+Transl+Med%22%5BJournal%5D)">Strategies for cardiac regeneration and repair.</a> Sci Transl Med. 6 (239), 239rv231(2014).</li>
<li>Writing Group, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+Disease+and+Stroke+Statistics-2016+Update%3A+A+Report+From+the+American+Heart+Association%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association.</a> Circulation. 133 (4), e38-e360 (2016).</li>
<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li>
<li>Song, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+repair+by+reprogramming+non-myocytes+with+cardiac+transcription+factors%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Heart repair by reprogramming non-myocytes with cardiac transcription factors.</a> Nature. 485 (7400), 599-604 (2012).</li>
<li>Ieda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+reprogramming+of+fibroblasts+into+functional+cardiomyocytes+by+defined+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.</a> Cell. 142 (3), 375-386 (2010).</li>
<li>Qian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+reprogramming+of+murine+cardiac+fibroblasts+into+induced+cardiomyocytes%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.</a> Nature. 485 (7400), 593-598 (2012).</li>
<li>Fu, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+reprogramming+of+human+fibroblasts+toward+a+cardiomyocyte-like+state%5BTitle%5D)+AND+%22Stem+Cell+Reports%22%5BJournal%5D)">Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state.</a> Stem Cell Reports. 1 (3), 235-247 (2013).</li>
<li>Zhou, H., Dickson, M. E., Kim, M. S., Bassel-Duby, R., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Akt1%2Fprotein+kinase+B+enhances+transcriptional+reprogramming+of+fibroblasts+to+functional+cardiomyocytes%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes.</a> Proc Natl Acad Sci U S A. 112 (38), 11864-11869 (2015).</li>
<li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bmi1+Is+a+Key+Epigenetic+Barrier+to+Direct+Cardiac+Reprogramming%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Bmi1 Is a Key Epigenetic Barrier to Direct Cardiac Reprogramming.</a> Cell Stem Cell. 18 (3), 382-395 (2016).</li>
<li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-efficiency+reprogramming+of+fibroblasts+into+cardiomyocytes+requires+suppression+of+pro-fibrotic+signalling%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling.</a> Nat Commun. 6, 8243(2015).</li>
<li>Miki, K., Yoshida, Y., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+steady+progress+on+direct+cardiac+reprogramming+toward+clinical+application%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Making steady progress on direct cardiac reprogramming toward clinical application.</a> Circ Res. 113 (1), 13-15 (2013).</li>
<li>Atkinson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anatomical+and+molecular+mapping+of+the+left+and+right+ventricular+His-Purkinje+conduction+networks%5BTitle%5D)+AND+%22J+Mol+Cell+Cardiol%22%5BJournal%5D)">Anatomical and molecular mapping of the left and right ventricular His-Purkinje conduction networks.</a> J Mol Cell Cardiol. 51 (5), 689-701 (2011).</li>
<li>Bootman, M. D., Smyrnias, I., Thul, R., Coombes, S., Roderick, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Atrial+cardiomyocyte+calcium+signalling%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">Atrial cardiomyocyte calcium signalling.</a> Biochim Biophys Acta. 1813 (5), 922-934 (2011).</li>
<li>Miquerol, L., Beyer, S., Kelly, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+the+mouse+ventricular+conduction+system%5BTitle%5D)+AND+%22Cardiovasc+Res%22%5BJournal%5D)">Establishment of the mouse ventricular conduction system.</a> Cardiovasc Res. 91 (2), 232-242 (2011).</li>
<li>Später, D., Hansson, E. M., Zangi, L., Chien, K. R. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/25406392">How to make a cardiomyocyte.</a> Development. 141 (23), 4418-4431 (2014).</li>
<li>Nam, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+diverse+cardiac+cell+types+by+reprogramming+fibroblasts+with+cardiac+transcription+factors%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors.</a> Development. 141 (22), 4267-4278 (2014).</li>
<li>Conner, D. A. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aConner%2C+DA+%22Current+Protocols+in+Molecular+Biology%22">Current Protocols in Molecular Biology</a>. , John Wiley & Sons, Inc. (2001).</li>
<li>Jozefczuk, J., Drews, K., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+of+mouse+embryonic+fibroblast+cells+suitable+for+culturing+human+embryonic+and+induced+pluripotent+stem+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells.</a> J Vis Exp. (64), (2012).</li>
<li>Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., Yee, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vesicular+stomatitis+virus+G+glycoprotein+pseudotyped+retroviral+vectors%3A+concentration+to+very+high+titer+and+efficient+gene+transfer+into+mammalian+and+nonmammalian+cells%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells.</a> Proc Natl Acad Sci U S A. 90 (17), 8033-8037 (1993).</li>
<li>Ichim, C. V., Wells, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+high-titer+viral+preparations+by+concentration+using+successive+rounds+of+ultracentrifugation%5BTitle%5D)+AND+%22J+Transl+Med%22%5BJournal%5D)">Generation of high-titer viral preparations by concentration using successive rounds of ultracentrifugation.</a> J Transl Med. 9, 137(2011).</li>
<li>Qian, L., Berry, E. C., Fu, J. D., Ieda, M., Srivastava, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reprogramming+of+mouse+fibroblasts+into+cardiomyocyte-like+cells+in+vitro%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro.</a> Nat Protoc. 8 (6), 1204-1215 (2013).</li>
<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

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 ...

assessing-cardiomyocyte-subtypes-following-transcription-factor

Research

JoVE Journal

Developmental Biology

7.4K Views.  UT Southwestern Medical Center. 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. 

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

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.

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 ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

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.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

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 first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

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.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

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

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

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

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.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

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 mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

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-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

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

Assessing Cardiac Reprogramming using High Content Imaging Analysis

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a reprogramming technique. Cardiac reprogramming provides a strategy to generate new cardiomyocytes. By introducing core cardiogenic transcription factors into fibroblasts; fibroblasts can be converted to iCMs without transition through the pluripotent stem cell state. However, the conversion rate of fibroblasts to iCMs still remains low. Accordingly, there have been numerous additional approaches to enhance cardiac reprogramming efficiency. Most of these studies assessed cardiac reprogramming efficiency using flow cytometry, while at the same time performed immunocytochemistry to visualize iCMs. Thus, at least two separate sets of reprogramming experiments are required to demonstrate the success of iCM reprogramming. In contrast, automated high content imaging analysis will provide both quantification and qualification of iCM reprogramming with a relatively small number of cells. With this method, it is possible to directly assess the quantity and quality of iCMs with a single reprogramming experiment. This approach will be able to facilitate future cardiac reprogramming studies that require large-scale reprogramming experiments such as screening genetic or pharmacological factors for enhancing reprogramming efficiency. In addition, the application of high content imaging analysis protocol is not limited to cardiac reprogramming. It can be applied to reprogramming of other cell lineages as well as any immunostaining experiments which need both quantification and visualization of immunostained cells.

We present a protocol to quantify directly reprogrammed induced cardiomyocyte-like cells (iCMs) in vitro using high content imaging analysis. This method allows us to quantify the efficiency of cardiac reprogramming in an automated manner and to directly visualize iCMs.

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Biology

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

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.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

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

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

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.

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

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

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including Mef2c, Gata4, Tbx5 (MGT), fibroblasts can be reprogrammed into induced cardiomyocytes (iCMs). These iCMs, when generated in situ in an infarcted heart, integrate electrically and mechanically with the surrounding myocardium, leading to a reduction in scar size and an improvement in heart function. Because of the relatively low reprogramming efficiency, purity, and quality of the iCMs, characterization of iCMs remains a challenge. The currently used methods in this field, including flow cytometry, immunocytochemistry, and qPCR, mainly focus on cardiac-specific gene and protein expression but not on the functional maturation of iCMs. Triggered by action potentials, the opening of voltage-gated calcium channels in cardiomyocytes leads to a rapid influx of calcium into the cell. Therefore, quantifying the rate of calcium influx is a promising method to evaluate cardiomyocyte function. Here, the protocol introduces a method to evaluate iCM function by calcium (Ca2+) flux. An &#945;MHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 mouse strain was established by crossing Tg(Myh6-cre)1Jmk/J (referred to as Myh6-Cre below) with Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as Rosa26A-Flox-Stop-Flox-GCaMP3 below) mice. Neonatal cardiac fibroblasts (NCFs) from P0-P2 neonatal mice were isolated and cultured in vitro, and a polycistronic construction of MGT was introduced to NCFs, which led to their reprogramming to iCMs. Because only successfully reprogrammed iCMs will express GCaMP3 reporter, the functional maturation of iCMs can be visually assessed by Ca2+ flux with fluorescence microscopy. Compared with un-reprogrammed NCFs, NCF-iCMs showed significant calcium transient flux and spontaneous contraction, similar to CMs. This protocol describes in detail the mouse strain establishment, isolation and selection of neonatal mice hearts, NCF isolation, production of retrovirus for cardiac reprogramming, iCM induction, the evaluation of iCM Ca2+ flux using our reporter line, and related statistical analysis and data presentation. It is expected that the methods described here will provide a valuable platform to assess the functional maturation of iCMs for cardiac reprogramming studies.

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as &#945;MHC-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.

Cardiac reprogramming has become a potentially promising therapy to repair a damaged heart. By introducing multiple transcription factors, including ...

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

Samenvatting

Meer video's verkennen