This research was supported by funds from the Boettcher Foundation&#39;s Webb-Waring Biomedical Research Program, American Heart Association Scientist Development Grant (13SDG17400031), University of Colorado Department of Medicine Outstanding Early Career Scholar Program, University of Colorado Division of Cardiology Barlow Nyle endowment, and NIH R01HL133230 (to K.S). A.S.R was supported by NIH/NCATS Colorado CTSA Grant Number TL1TR001081 and a pre-doctoral fellowship from the University of Colorado Consortium for Fibrosis Research &#38; Translation (CFReT). This research was also supported by the Cancer Center Support Grant (P30CA046934), the Skin Diseases Research Cores Grant (P30AR057212), and the Flow Cytometry Core at the University of Colorado Anschutz Medical Campus.

All experiments requiring animals were approved by the Institutional Animal Care and Use Committee at the UC Denver Anschutz Medical Campus.
1. Isolation of MEFs
<ol>
	<li>Purchase C57BL/6 pregnant mice at E13. Ship overnight.</li>
	<li>Euthanize the mother according to approved IACUC protocols (ex: ~1.3 L/min CO2 until animal appears dead followed by cervical dislocation)</li>
	<li>Spray the mother with 70% ethanol and open abdominal cavity. Remove the uterine horn containing embryos and place in a 10 cm dish with sterile PBS.</li>
	<li>Make an incision in the embryo sac to release embryo. Transfer embryo to clean 10 cm dish with sterile PBS. Rinse thoroughly.</li>
	<li>Cut the body below the liver and discard the head and upper body. Remove internal organs and discard. Transfer the remaining tissue to a clean 10 cm dish with sterile PBS and transfer the plate to a Biosafety Level 1 or 2 Cabinet. Combine tissues from all embryos in a clean, dry 10 cm dish and mince into fine pieces.</li>
	<li>Add 6 mL of 0.25% trypsin/1 mM EDTA to minced embryos. Triturate several times by pipette to break up tissue. Incubate for 40 min at 37 &#730;C with 5% CO2.</li>
	<li>Resuspend cells in growth medium (Table 1). The volume of media is adjusted based on the number of embryos harvested. In general, use a ratio of approximately 25 mL growth media per 1.2 embryos.</li>
	<li>Plate 25 mL of resuspended cells in a 15 cm dish. After 24 h, aspirate the media and add 25 mL of fresh growth medium to each plate.</li>
	<li>After 72 h, add 2 mL of 0.25% trypsin/1 mM EDTA to each 15 cm dish. When cells detach from the culture dish, inactivate trypsin with 15 mL growth medium and collect cells in 50 mL polystyrene conical tubes. Centrifuge cells for 3 min at 160 x g at room temperature. Resuspend the cell pellet in growth medium and filter through a 70 &#181;m pore cell strainer.</li>
	<li>Count cells using a hemocytometer and freeze MEFs in 2 million cells aliquots in freeze medium (10% DMSO, 25% FBS in Dulbecco&#39;s Modified Eagle Medium (DMEM)/High glucose) at -80 &#176;C. Transfer cells to liquid nitrogen after 24 h and store until ready to use. 
	NOTE: Global gene expression in MEFs was profiled isolated using this protocol by RNA-sequencing (RNA-seq) to ensure cell authentication. RNA-seq data from ENCODE MEF cells were downloaded from NIH Gene Expression Omnibus (GEO) with accession number GSE93454. RNA-seq gene expression data for the MEFs used by us were deposited in NIH GEO with accession number GSE71405. These two data sets were merged according to common gene symbols. The expression data were then log-transformed and a scatterplot of the expression data for our MEFs and ENCODE MEFs were generated and fit with a linear regression line (Figure 1A). MEFs isolated using this protocol are molecularly similar to those isolated by other labs.</li>
</ol>
2. Production of Retrovirus and Transduction of MEFs
NOTE: All steps in this section must be carried out in a Biosafety Level 2 Cabinet. Since this protocol utilizes retroviral transduction, ensure that safety precautions are taken including treating all waste containing viral media with 10% bleach for at least 20 min. Refer to Figure 1B for a timeline for reprogramming.
<ol>
	<li>Production of Retrovirus using the Platinum E (PE) cell line
	<ol>
		<li>Maintain commercially available PE cells in PE medium (Table 1) containing blasticidin and puromycin selection markers to ensure expression of gag-pol and env genes for efficient retroviral particle production36. Passage cells at 1:4 or 1:5 every two days. Take care to prevent overcrowding of cells as this can reduce retroviral production yields.</li>
		<li>On the day -1, seed approximately 5 x 106 cells per 10 cm dish in the growth medium (Table 1) 16-20 h prior to transfection (see Table 2 for plating densities for other standard cell culture dish sizes). Gently rock dishes back and forth several times and carefully place in a 37 &#176;C, 5% CO2 incubator to ensure even plating distribution (see Figure 1C for optimal plating density prior to transfection). 
		NOTE: Take care to plate PE cells in the medium free of selection markers prior to transfection to ensure medium containing generated retroviral particles does not kill MEFs.</li>
		<li>On the day 0, prepare DNA for transfection. For 10 cm dish transfection, add 2 &#181;g of each reprogramming factor &#8212; GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) &#8212; to a 1.5 mL tube. 
		NOTE: Refer to Table 2 to scale the amount of DNA needed to transfect other standard cell culture dish sizes.</li>
		<li>In a separate 1.5 mL tube, add 360 &#181;L reduced serum media. Then add 36 &#181;L transfection reagent directly to reduced serum media. Gently flick the tube and incubate at room temperature for 5 min. 
		NOTE: To avoid reduced transfection efficiency, carefully add transfection reagent directly to the reduced serum media.</li>
		<li>Add reduced serum media/transfection reagent mixture to GHMT2m DNA cocktail. Gently flick the tube and incubate at room temperature for 15 min. Do not vortex.</li>
		<li>Add the reaction mixture dropwise to PE cells, gently swirl media for 10-15 s, and place in a 37 &#176;C, 5% CO2 incubator. 
		NOTE: According to the manufacturer, PE cells generate an average titer of 106 to 107 infectious units/mL. This protocol generates approximately 2 x 106 infections units/mL by 24 h and 6 x 106 infections units/mL by 48 h for an average MOI of 4. Transfection of GFP/DsRed may also be used as a surrogate for transfection/transduction efficiency if desired; transfection efficiency is expected to exceed 90% by 48 h (Figure 1C).</li>
	</ol>
	</li>
	<li>Transduction of MEFs
	<ol>
		<li>On the Day 0, coat dishes with collagen and place in a 37 &#176;C, 5% CO2 incubator for at least 2 h.</li>
		<li>Remove MEFs from liquid nitrogen and thaw. Plate 0.2 x 106 cells per 60 mm dish in the growth medium. Gently rock plates to ensure even plating distribution and place in the incubator (see Figure 1C for optimal plating density and Table 2 for plating densities for other standard cell culture dish sizes). 
		NOTE: These plating densities have been tested over multiple batches of isolated MEFs; unless MEFs display significantly compromised viability, adjusting cell seeding densities is not necessary.</li>
		<li>On the day 1, 24 h post-transfection, use a 30 mL syringe to collect retroviral medium generated by PE cells. Pass the medium through a 0.45 &#181;m pore-size filter into a 50 mL conical tube. Add hexadimethrine bromide transfection reagent to a final concentration of 6 &#181;g/mL. Carefully add 10 mL fresh growth medium to PE cells by pipetting media onto the wall of the culture dish to prevent displacement of cells.</li>
		<li>Aspirate the medium from MEFs and add 4 mL of freshly harvested retroviral medium to each dish. Return MEFs to the incubator. Add 10% bleach to all materials in contact with the viral medium to neutralize retroviral particles.</li>
		<li>On the day 2, 48 h post-transfection, repeat steps 2.2.2 and 2.2.3. PE cells are discarded following the 2nd collection of the retroviral medium. 
		NOTE: Add 10% bleach to discarded PE cells for at least 20 min to neutralize unwanted retrovirus.</li>
		<li>On the day 3, aspirate medium from MEFs and replenish with 4 mL iCM medium (Table 1) containing 0.5 &#181;M A-83-01, a TGF-&#946; type I receptor inhibitor. Replenish iCM medium containing A-83-01 every 2 days. 
		NOTE: If using GFP as a transduction control, the expression is expected to exceed 90% by this time point (Figure 1C).</li>
	</ol>
	</li>
</ol>
3. Flow Cytometry for Cardiac Markers
<ol>
	<li>Aspirate medium from Day 9 reprogrammed MEFs and wash (1x) with 1x PBS to remove dead cells and debris.</li>
	<li>Add 1 mL 0.25% Trypsin/EDTA for 5 min at 37 &#730;C. Check cells using a light microscope and shake dish; if cells remain attached, incubate 5 more min at 37 &#730;C until cells detach.</li>
	<li>Wash cells in 1x PBS containing 5% FBS and filter through a 70 &#181;m pore cell strainer.</li>
	<li>Centrifuge at 160 x g for 3 min at room temperature and aspirate liquid from the pellet. 
	NOTE: To increase the cell yield, leave approximately 50 &#181;L liquid above the cell pellet.</li>
	<li>Wash cells with 500 &#181;L 1x PBS containing 1% BSA.</li>
	<li>Fix cells with 0.2 mL/1 million cells fixation solution for 20 min on ice.</li>
	<li>Add 1 mL of 1x ice-cold perm/wash buffer. 
	NOTE: The manufacturer&#39;s solution is 10x.</li>
	<li>Centrifuge cells at 160 x g for 5 min at 4 &#176;C and aspirate supernatant.</li>
	<li>Repeat steps 3.7 and 3.8 an additional time. After the 2nd wash, proceed directly to next step or keep cells at 4 &#176;C overnight.</li>
	<li>Block with 100 &#181;L of 5% goat serum and donkey serum in 1x perm/wash buffer for 30 min at room temperature.</li>
	<li>Add 100 &#181;L of diluted primary antibody. 
	NOTE: For this protocol, cells were labeled with mouse Troponin T (1:200) and mouse &#945;-actinin (1:200) to quantify the percentage of cells positive for key components of the cardiac sarcomere. Incubate primary antibody for 1 h at room temperature.</li>
	<li>Centrifuge cells at 160 x g for 5 m at 4 &#176;C, aspirate supernatant, and wash 2x with ice-cold perm/wash buffer.</li>
	<li>Add 100 &#181;L of diluted secondary antibody. 
	NOTE: For this protocol, cells were labeled with anti-mouse 647 nm secondary antibody (1:200). Incubate secondary antibody for 45 min at room temperature on an end-over-end tube rotator. Protect cells from light by wrapping tubes in foil.</li>
	<li>Centrifuge cells at 160 x g for 5 min at 4 &#176;C, aspirate supernatant, and wash 2x with ice-cold perm/wash buffer.</li>
	<li>Add 1 mL of 1% BSA in PBS and perform FACS immediately. 
	NOTE: Generate a forward scatter vs side scatter plot to gate viable cells first when performing FACS. Alternatively, use a commercially available live/dead cell stain prior to fixing cells to identify live and dead cell populations.</li>
</ol>
4. Immunostaining of Reprogrammed MEFs
<ol>
	<li>Aspirate medium from day 14 reprogrammed MEFs and wash (1x) with 1 mL 1x ice-cold PBS. 
	NOTE: For immunostaining, reprogramming was performed in 12 well plates to minimize antibody solution volumes. 24 well plates may also be used; simply half all following volumes.</li>
	<li>Aspirate and fix cells with 500 &#181;L 2% paraformaldehyde for 10 min at room temperature.</li>
	<li>Aspirate and wash cells 3 times with 1 mL 1x PBS (5 min per wash at room temperature).</li>
	<li>Permeabilize cells with 500 &#181;L 0.2% permeabilization reagent in PBS for 15 min at room temperature.</li>
	<li>Aspirate and block in 500 &#181;L 10% horse serum in PBS for 30 min at room temperature.</li>
	<li>Aspirate and incubate with 500 &#181;L of the diluted primary antibody for 1 h at room temperature. 
	NOTE: For this protocol, cells were labeled with mouse Troponin T or &#945;-actinin (each 1:400) and co-labeled with Connexin 43 (1:400) or Troponin I (1:400).</li>
	<li>Aspirate primary antibody and wash three times with 1 mL 1x PBS (5 min per wash at room temperature).</li>
	<li>Aspirate and incubate with 500 &#181;L diluted secondary antibody for 1 hour at room temperature. 
	NOTE: For this protocol, cells were labeled with anti-mouse 555 nm secondary antibody (1:800) to recognize Troponin T or &#945;-actinin and anti-rabbit 488 nm secondary antibody (1:800) to recognize Connexin 43 or Troponin I. Additionally, nuclei were stained with Hoechst (1:10,000). Protect cells from light by incubating in the dark.</li>
	<li>Aspirate the secondary antibody and wash three times with 1 mL 1x PBS (5 min per wash at room temperature). Wrap the plate in foil to protect cells from light and store at 4 &#176;C.</li>
	<li>Image using three-channel epifluorescence microscopy.</li>
</ol>
5. Calcium Imaging
NOTE: If imaging at 10X magnification, standard cell culture dishes and plates are suitable for calcium imaging. Imaging at higher magnification requires that cells be plated on glass coverslips or glass bottom dishes.
<ol>
	<li>Aspirate medium and add 2 mL (for a 6 well plate) modified Tyrode&#39;s solution (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, 0.1% BSA, and 1% pyruvate, pH 7.4) containing 5 &#181;M Fura-2 AM and 0.1% Pluronic F-127 to beating (D14 to D20) iCMs. Incubate for 30 minutes at 37 &#176;C. 
	NOTE: Protect cells from light to prevent quenching of fluorescent indicator.</li>
	<li>Wash cells in 2 mL Tyrode&#39;s solution for 30 min at room temperature to allow de-esterification of Fura-2 AM.</li>
	<li>Image with spinning disk confocal microscopy at 340 nm and 380 nm using Slidebook 5.5 Ca2+ imaging software. Perform the imaging at room temperature.</li>
	<li>Calcium transients are calculated using the ratio of fluorescence intensity at 340 nm over the fluorescence intensity at 380 nm.</li>
	<li>If desired, treat iCMs with 1 or 2 &#181;M isoproterenol or 10 &#181;M Nifedipine to manipulate calcium handling after imaging baseline calcium transients. Add compounds locally to cells and image.</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+TGF&#946;+signaling+increases+direct+conversion+of+fibroblasts+to+induced+cardiomyocytes.">Inhibition of TGF&#946; signaling increases direct conversion of fibroblasts to induced cardiomyocytes.</a> PLoS One. 9 (2), e89678 (2014).</li><li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmi1+Is+a+Key+Epigenetic+Barrier+to+Direct+Cardiac+Reprogramming.">Bmi1 Is a Key Epigenetic Barrier to Direct Cardiac Reprogramming.</a> Cell Stem Cell. 18 (3), 382-395 (2016).</li><li>Christoforou, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors+MYOCD,+SRF,+Mesp1+and+SMARCD3+enhance+the+cardio-inducing+effect+of+GATA4,+TBX5,+and+MEF2C+during+direct+cellular+reprogramming.">Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 enhance the cardio-inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming.</a> PLoS One. 8 (5), e63577 (2013).</li><li>Jayawardena, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+induced+cardiac+reprogramming+in+vivo:+evidence+for+mature+cardiac+myocytes+and+improved+cardiac+function.">MicroRNA induced cardiac reprogramming in vivo: evidence for mature cardiac myocytes and improved cardiac function.</a> Circ Res. 116 (3), 418-424 (2015).</li><li>Jayawardena, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-mediated+in+vitro+and+in+vivo+direct+reprogramming+of+cardiac+fibroblasts+to+cardiomyocytes.">MicroRNA-mediated in vitro and in vivo direct reprogramming of cardiac fibroblasts to cardiomyocytes.</a> Circ Res. 110 (11), 1465-1473 (2012).</li><li>Cao, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+human+fibroblasts+into+functional+cardiomyocytes+by+small+molecules.">Conversion of human fibroblasts into functional cardiomyocytes by small molecules.</a> Science. 352 (6290), 1216-1220 (2016).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+reprogramming+of+fibroblasts+into+cardiomyocytes+requires+suppression+of+pro-fibrotic+signalling.">High-efficiency reprogramming of fibroblasts into cardiomyocytes requires suppression of pro-fibrotic signalling.</a> Nature Communications. 6, 1-15 (2015).</li><li>Morita, S., Kojima, T., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plat-E:+an+efficient+and+stable+system+for+transient+packaging+of+retroviruses.">Plat-E: an efficient and stable system for transient packaging of retroviruses.</a> Gene Therapy. 7, 1063-1066 (2000).</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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt1/protein+kinase+B+enhances+reprogramming+of+fibroblasts+to+functional+cardiomyocytes.">Akt1/protein kinase B enhances reprogramming of fibroblasts to functional cardiomyocytes.</a> Proc Natl Acad Sci. 112 (38), 11864-11869 (2015).</li><li>Zhou, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZFN281+enhances+cardiac+reprogramming+by+modulating+cardiac+and+inflammatory+gene+expression.">ZFN281 enhances cardiac reprogramming by modulating cardiac and inflammatory gene expression.</a> Genes &amp; Dev. 31, 1770-1783 (2017).</li><li>Mohamed, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+Enhancement+of+In+Vitro+and+In+Vivo+Direct+Cardiac+Reprogramming.">Chemical Enhancement of In Vitro and In Vivo Direct Cardiac Reprogramming.</a> Circulation. 135, 978-995 (2017).</li><li>Qian, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+reprogramming+of+murine+cardiac+fibroblasts+into+induced+cardiomyocytes.">In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes.</a> Nature. 485, 593-598 (2012).</li><li>Mathison, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+reprogramming+to+transdifferentiate+fibroblasts+into+cardiomyocytes+using+adenoviral+vectors:+Implications+for+clinical+myocardial+regeneration.">In situ reprogramming to transdifferentiate fibroblasts into cardiomyocytes using adenoviral vectors: Implications for clinical myocardial regeneration.</a> J Thorac Cardiovasc Surg. , 329-339 (2017).</li></ol>

The authors have nothing to disclose.

The present study outlines a high-efficiency strategy to directly reprogram fibroblasts into functional iCMs via delivery of GHMT2m reprogramming factors combined with suppression of pro-fibrotic signaling pathways. Using flow cytometry, immunofluorescent imaging, calcium imaging, and beating cell counts, we show the majority of cells in this protocol undergo successful reprogramming and adopt CM lineage fate. We have previously shown that the addition of anti-fibrotic compounds including the TGF-&#946; type I receptor i...

Ischemic heart disease is a leading cause of death in the United States1. Approximately 800,000 Americans experience a first or recurrent myocardial infarction (MI) per year1. Following MI, the death of cardiomyocytes (CMs) and cardiac fibrosis, deposited by activated cardiac fibroblasts, impair heart function2,3. Progression of heart failure following MI is largely irreversible due to the poor regenerative capacity of adult CMs4,5. While current clinical therapies slow disease progression and decrease risk of future cardiac events6,7,8,9, no therapies reverse disease progression due to the inability to regenerate CMs post-infarction10. Novel cell therapies are emerging to treat patients following MI. Disappointingly, clinical trials delivering stem cells to the heart following MI thus far have shown inconclusive regenerative potential11,12,13,14,15,16,17,18.
The generation of human-derived induced pluripotent stem cells (hiPSCs) from fibroblasts by overexpression of four transcription factors, first demonstrated by Takahashi &#38; Yamanaka, opened the door to new breakthroughs in cell therapy19. These cells can differentiate into all three germ layers19, and several highly efficient methods for generating large numbers of CMs have been previously shown20,21. HiPSC-derived CMs (hiPS-CMs) offers a powerful platform to study cardiomyogenesis and may have important implications for repairing the heart following injury. However, hiPS-CMs currently face translational hurdles due to concerns of teratoma formation22, and their immature nature may be pro-arrhythmogenic23. Reprogramming fibroblasts into hiPSCs sparked interest in directly reprogramming fibroblasts into other cell types. Ieda et al. demonstrated that overexpression of GATA4, Mef2c, and Tbx5 (GMT) in fibroblasts results in direct reprogramming to cardiac lineage, albeit at low efficiency24. Reprogramming efficiency was improved with the addition of Hand2 (GHMT)25. Since these early studies, many publications have demonstrated that altering the reprogramming factor cocktail with additional transcription factors26,27,28,29, chromatin modifiers30,31, microRNAs32,33, or small molecules34 leads to improved reprogramming efficiency and/or maturation of induced cardiomyocyte-like cells (iCMs).
Here we provide a detailed protocol to generate iCMs from mouse embryonic fibroblasts (MEFs) with high efficiency. We previously showed that the GHMT cocktail is significantly improved with the addition of miR-1 and miR-133 (GHMT2m) and is further improved when pro-fibrotic signaling pathways including transforming growth factor &#946; (TGF-&#946;) signaling or Rho-associated protein kinase (ROCK) signaling pathways are inhibited35. Using this protocol, we show that approximately 60% of cells express cardiac Troponin T (cTnT), approximately 50% express &#945;-actinin, and a high number of beating cells can be observed as early as Day 11 following transduction of reprogramming factors and treatment with the TGF-&#946; type I receptor inhibitor A-83-01. Furthermore, these iCMs express gap junction proteins including connexin 43 and exhibit spontaneous contraction and calcium transients. This marked improvement in reprogramming efficiency compared to earlier studies demonstrates the potential to regenerate CMs from endogenous cell populations that remain in the heart post-infarction.

<table><tbody><tr><td>C57BL/6 Mice</td><td>Charles River&#039;s Laboratory</td><td>027</td><td>For MEF isolation</td></tr><tr><td>Platinum E (PE) Cells</td><td>Cell Biolabs, INC</td><td>RV-101</td><td>For retrovirus production</td></tr><tr><td>DMEM High Glucose</td><td>Gibco</td><td>SH30022.FS</td><td>Component of iCM, PE, and Growth media</td></tr><tr><td>Medium 199</td><td>Life Technologies</td><td>11150-059</td><td>Component of iCM media</td></tr><tr><td>Fetal Bovine Serum</td><td>Gemini</td><td>100106</td><td>Component of iCM, PE, and Growth media</td></tr><tr><td>Donor Horse Serum</td><td>Gemini</td><td>100508 500</td><td>Component of iCM media</td></tr><tr><td>MEM Essential Amino Acids, 50X</td><td>Life Technologies</td><td>11130051</td><td>Component of iCM media</td></tr><tr><td>Sodium Pyruvate Solution, 100X</td><td>Life Technologies</td><td>11360070</td><td>Component of iCM media and for calcium imaging</td></tr><tr><td>MEM Non-Essential Amino Acids, 100X</td><td>Life Technologies</td><td>11140050</td><td>Component of iCM media</td></tr><tr><td>MEM Vitamin Solution, 100X</td><td>Life Technologies</td><td>11120-052</td><td>Component of iCM media</td></tr><tr><td>Insulin-Transferrin-Selenium</td><td>Gibco</td><td>41400045</td><td>Component of iCM media</td></tr><tr><td>B27</td><td>Gibco</td><td>17504-044</td><td>Component of iCM media</td></tr><tr><td>Penicilin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td>Component of iCM, PE, and Growth media</td></tr><tr><td>GlutaMAX (L-Glutamine Supplement)</td><td>Gibco</td><td>35050-061</td><td>Component of iCM, PE, and Growth media</td></tr><tr><td>Blasticidin-HCl</td><td>Life Technologies</td><td>A11139-03</td><td>Component of PE media</td></tr><tr><td>Puromycin dihydrochloride</td><td>Life Technologies</td><td>A11138-03</td><td>Component of PE media</td></tr><tr><td>0.25% Trypsin/EDTA</td><td>Gibco</td><td>25200-056</td><td>For detaching cells from culture dishes</td></tr><tr><td>A-83-01</td><td>R&amp;amp;D Systems - Tocris</td><td>2939/10</td><td>Treat cells to inhibit TGF-&amp;beta; signaling - promotes high efficiecy reprogramming. Use at 0.5 &amp;micro;M</td></tr><tr><td>DMSO</td><td>Thermo Scientific</td><td>85190</td><td>For dilution and storage of A-83-01 and component of Freeze Medium</td></tr><tr><td>SureCoat</td><td>Cellutron</td><td>SC-9035</td><td>For coating dishes to plate MEFs</td></tr><tr><td>FuGENE 6 Transfection Reagent</td><td>Promega</td><td>E2692</td><td>Transfection Reagent</td></tr><tr><td>Opti-MEM Reduced Serum Media</td><td>Gibco</td><td>11058-021</td><td>Transfection Reagent</td></tr><tr><td>pBabe-X Myc-GATA4</td><td></td><td></td><td>Plasmid containing reprogramming factor</td></tr><tr><td>pBabe-X Myc-Hand2</td><td></td><td></td><td>Plasmid containing reprogramming factor</td></tr><tr><td>pBabe-X Myc-Mef2c</td><td></td><td></td><td>Plasmid containing reprogramming factor</td></tr><tr><td>pBabe-X Myc-Tbx5</td><td></td><td></td><td>Plasmid containing reprogramming factor</td></tr><tr><td>pBabe-X miR-1</td><td></td><td></td><td>Plasmid containing reprogramming factor</td></tr><tr><td>pBabe-X miR-133</td><td></td><td></td><td>Plasmid containing reprogramming factor</td></tr><tr><td>pBabe-X GFP</td><td></td><td></td><td>Plasmid containing reprogramming factor</td></tr><tr><td>Polybrene (Hexadimethrine bromide)</td><td>Sigma</td><td>H9268-5G</td><td>For viral induction. Use at a concentration of 6 &amp;micro;g/mL</td></tr><tr><td>Vacuum Filter + bottles (0.22 &amp;micro;m pores)</td><td>Nalgene</td><td>&amp;nbsp;569-0020&amp;nbsp;</td><td>For filtering media</td></tr><tr><td>Syringes</td><td>Bd Vacutainer Labware&amp;nbsp;</td><td>309654</td><td>For viral filtration</td></tr><tr><td>0.45 &amp;micro;m Filters</td><td>Celltreat</td><td>229749</td><td>For viral filtration</td></tr><tr><td>70 &amp;micro;m cell strainers</td><td>Falcon&amp;nbsp;</td><td>352350</td><td>For MEF isolation and Flow Cytometry</td></tr><tr><td>Cytofix/Cytoperm Solution</td><td>BD</td><td>554722</td><td>For fixation and permeabilization of cells for flow cytometry</td></tr><tr><td>perm/wash buffer&amp;nbsp;</td><td>BD</td><td>554723</td><td>For washing cells for flow cytometry</td></tr><tr><td>DPBS 1X</td><td>Gibco</td><td>14190-250</td><td>For washing cells</td></tr><tr><td>Bovine Serum Albumin</td><td>VWR</td><td>0332-100g</td><td>For flow cytometry and calcium imaging</td></tr><tr><td>Goat Serum</td><td>Sigma&amp;nbsp;</td><td>G9023</td><td>For blocking cells for Flow Cytometry</td></tr><tr><td>Donkey Serum</td><td>Sigma</td><td>D9663-10mg</td><td>For blocking cells for Flow Cytometry</td></tr><tr><td>Mouse Troponin T</td><td>Thermo Scientific</td><td>ms-295-p</td><td>1:400 IF, 1:200 Flow Cytometry</td></tr><tr><td>Mouse &amp;alpha;-actinin</td><td>Sigma</td><td>A7811L</td><td>1:400 IF, 1:200 Flow Cytometry</td></tr><tr><td>Rabbit Connexin 43</td><td>Sigma&amp;nbsp;</td><td>C6219</td><td>1:400 IF</td></tr><tr><td>Rabbit Troponin I</td><td>PhosphoSolutions</td><td>2010-TNI</td><td>1:400 IF</td></tr><tr><td>Hoechst</td><td>Life Technologies</td><td>62249</td><td>1:10000 IF</td></tr><tr><td>Alexa 488, rabbit</td><td>Life Technologies</td><td>A-11034</td><td>1:800 IF</td></tr><tr><td>Alexa 555, mouse</td><td>Life Technologies</td><td>A-21422</td><td>1:800 IF</td></tr><tr><td>Alexa 647, mouse</td><td>Life Technologies</td><td>A-31571</td><td>1:200 Flow Cytometry</td></tr><tr><td>27-color ZE5 Flow Cytometer&amp;nbsp;</td><td>Bio-RAD</td><td></td><td>For FACS</td></tr><tr><td>Paraformaldehyde</td><td>sigma</td><td>P6148-500mg</td><td>For fixing cells for IF</td></tr><tr><td>Triton X-100</td><td>Promega</td><td>H5142</td><td>For permeabilization of cells for IF</td></tr><tr><td>EVOS&amp;trade; FL Color Imaging System</td><td>Thermo Scientific</td><td>AMEFC4300</td><td>For IF</td></tr><tr><td>NaCl</td><td>RPI</td><td>S23020-5000</td><td>For calcium imaging</td></tr><tr><td>KCl</td><td>VWR</td><td>395</td><td>For calcium imaging</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>C614-500</td><td>For calcium imaging</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>VWR</td><td>97061-352</td><td>For calcium imaging</td></tr><tr><td>glucose</td><td>sigma</td><td>G7528-250g</td><td>For calcium imaging</td></tr><tr><td>HEPES</td><td>sigma</td><td>H4034-500g</td><td>For calcium imaging</td></tr><tr><td>Fura-2 AM</td><td>Life Technologies</td><td>F1221</td><td>For calcium imaging</td></tr><tr><td>Fluronic F-127</td><td>Sigma</td><td>P2443-250g</td><td>For calcium imaging</td></tr><tr><td>Nifedipine</td><td>Sigma</td><td>N7634-1G</td><td>For disruption calcium transients in iCMs - use at 10 &amp;micro;M</td></tr><tr><td>Isoproterenol</td><td>sigma</td><td>I6504-1g</td><td>For increasing number of calcium transients in iCMs - use at 1-2 &amp;micro;M</td></tr><tr><td>Marianas Spinning Disk Confocal microscope</td><td>3i</td><td></td><td>For calcium imaging</td></tr><tr><td>ethanol</td><td>Decon Laboratories</td><td>2801</td><td></td></tr><tr><td>bleach</td><td>Clorox</td><td></td><td></td></tr><tr><td>50 mL conical tubes</td><td>GREINER BIO-ONE</td><td>227261</td><td></td></tr><tr><td>15 mL conical tubes</td><td>GREINER BIO-ONE</td><td>188271</td><td></td></tr><tr><td>15 cm cell culture dishes</td><td>Falcon</td><td>353025</td><td></td></tr><tr><td>10 cm cell culture dishes</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>60 mm cell culture dishes</td><td>GREINER BIO-ONE</td><td>628160</td><td></td></tr><tr><td>6 well cell culture plates</td><td>GREINER BIO-ONE</td><td>657160&amp;nbsp;</td><td></td></tr><tr><td>12 well cell culture plates</td><td>GREINER BIO-ONE</td><td>665180&amp;nbsp;</td><td></td></tr><tr><td>24 well cell culture plates</td><td>GREINER BIO-ONE</td><td>662160&amp;nbsp;</td><td></td></tr></tbody></table>

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.

Using the reprogramming strategy outlined above and in Figure 1B, we generated iCMs with approximately 70% of cells expressing cardiac Troponin T and approximately 55% of cells expressing cardiac &#945;-actinin, quantified by flow cytometry at Day 9 following transduction of GHMT2m (Figure 2A and B). Additionally, the majority of cells express cardiac Troponin T, Troponin I, and cardiac &#945;-actinin as well as ...

Watch this Scientific Journal Video about Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes at JoVE.com

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

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 ...

suppression-pro-fibrotic-signaling-potentiates-factor-mediated

Research

JoVE Journal

Developmental Biology

7.2K Views.  University of Colorado Anschutz Medical Campus. Hier presenteren we een robuuste methode om te herprogrammeren primaire embryonale fibroblasten in functionele cardiomyocytes via overexpressie van GATA4, Hand2, Mef2c, Tbx5, miR-1 en miR-133 (GHMT2m) naast remming van TGF-β signalering. Ons protocol genereert kloppend cardiomyocytes zo spoedig 7 dagen na transductie met korting tot 60% efficiëntie.

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

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 ...

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 ...

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.

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 ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Biochemistry

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

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 ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Biology

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 ...

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 ...

Medicine

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

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.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

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

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