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

The protocol outlined here uses neonatal mice. Animal care and experiments are performed in accordance with the guidelines established by The Division of Laboratory Animal Medicine (DLAM) at University of North Carolina, Chapel Hill.

1. Preparation of Buffers and Media

1. Prepare fibroblast (FB) medium: Supplement 500 ml IMDM media with 100 ml Fetal Bovine Serum (FBS) and 6 ml penicillin/streptomycin.
2. Prepare iCM medium: Supplement 400 ml DMEM media with 100 ml M199 media and 50 ml FBS.
3. Prepare B27 medium: Supplement 490 ml RPMI1640 media with 10 ml B27 media.
4. Prepare Plat-E culture medium: Supplement 500 ml DMEM media with 50 ml FBS, 5 ml penicillin/streptomycin and 5 ml Non-essential amino acids.
5. Prepare Plat-E transfection medium: Supplement 500 ml DMEM media with 50 ml FBS and 5 ml Non-essential amino acids.
6. Prepare Gelatin coated 24 well plate: Add 0.5 ml 0.1% gelatin solution to a well of 24 well plate, incubate at 37 °C for at least 10 min and aspirate right before use.
7. Prepare FACS labeling buffer: Supplement 500 ml DPBS with 10 ml FBS and 2 ml EDTA (0.5 M) to reach a final concentration of 2 mM. Pass through a 0.22 µm filter and store at 4 °C.
8. Prepare MACS sorting buffer: Supplement 500 ml DPBS with 2.5 g BSA and 2 ml EDTA (0.5 M) to reach a final concentration of 2 mM. Pass through a 0.22 µm filter and store at 4 °C.
9. Prepare 2x HBS buffer for transfection: supplement 500 ml HEPES buffer (50 mM) with 280 mM NaCl (8.1816 g), 10 mM KCl (0.37275 g), 1.5 mM Na₂HPO₄ (0.10647 g), 12 mM Glucose (1.08096 g). Add around 650 µl 10 N NaOH to adjust pH to 7.05-7.12. Filter through a 0.22 µm pore filter and store at 4 °C.
10. Prepare 2.5 M CaCl₂ solution for transfection: Add 18.376 g CaCl₂ to 50 ml ddH₂O. Filter through a 0.22 µm pore filter and store at 4 °C.
11. Prepare ICC (immunocytochemistry) fixation buffer: Add 5 ml paraformaldehyde (PFA, 32%) to 35 ml DPBS to reach a final concentration of 4%.
12. Prepare ICC permeabilization buffer: Add 0.1 ml Triton to 100 ml DPBS to reach a final concentration of 0.1%.
13. Prepare ICC blocking buffer: Add 5 g Bovine serum albumin (BSA) to 100 ml DPBS to reach a final concentration of 5%.
14. Prepare ICC staining buffer: Add 1 g BSA to 100 ml DPBS to reach a final concentration of 1%.

2. Generation of Neonatal Mouse Cardiac Fibroblasts

1. Generate cardiac fibroblasts from explant culture method
   1. Clean neonatal αMHC-GFP transgenic mouse (P1 to P2) with 75% ethanol. Cut the head off with sterile scissors. Then make a horizontal incision from under one armpit to the other. Use a sterile blunt and bent forceps to dissect out the heart and place it in one well of the 24 well plate containing ice-cold PBS buffer.
      1. Alternatively, squeeze out the heart after the horizontal incision.
   2. Check GFP expression in heart by fluorescent microscope. Since the GFP expression is driven by αMHC promoter that is a cardiac specific promoter, the heart from transgenic mouse should be in green¹⁵, while the negative heart is dim in any fluorescence.
   3. Take 3-4 GFP positive hearts into a 60 mm center well culture dish and mince them into small pieces less than 1 mm³ in size by sterile scissors and forceps.
   4. Place 3-4 minced hearts into one 10 cm dish with 2 ml FB media. Let the tissues settle down for 3 hr.
   5. Slowly add 8 ml pre-warmed FB media to the dishes containing heart tissues. Do not disturb the tissues for three days.
   6. Replace the media every three days.
   7. On day 7, aspirate the culture medium and wash cells with DPBS. Add 3 ml 0.05% trypsin to each plate and digest at 37 °C for 5 min.
   8. Add 5 ml FB media to quench trypsin. Gently detach the cells with a cell scraper. Pipette the media up and down to further mechanically dissociate the tissue.
   9. Collect cells and filter through 40 µm cell strainers to avoid contamination of heart tissue fragments, and then pellet cells by spinning at 200 x g for 5 min.
   10. Wash cells once with MACS buffer and cells are ready for sorting.
2. Generate cardiac fibroblasts from enzyme digestion method
   1. Harvest GFP positive hearts as 2.1.1. Transfer all the hearts into a 10 cm dish with 10 ml ice-cold DPBS.
   2. Squeeze ventricles with sterile forceps to remove blood and rinse once with ice-cold DPBS.
   3. Trim the hearts to be free of other tissues and fat.
   4. Cut each heart into roughly 4 pieces that are still loosely connected.
   5. If less than 20 hearts, transfer the hearts into a 15 ml conical tube with 8 ml warm 0.05% Trypsin-EDTA, and incubate at 37 °C for 15 min.
      1. If there are 20-30 hearts, transfer the hearts into a 50 ml conical tube with 10 ml warm 0.05% Trypsin-EDTA, and incubate at 37 °C for 15 min.
   6. Discard the trypsin and add 5 ml (for less than 20 hearts) or 10 ml (for 20-30 hearts) warm type II collagenase (0.5 mg/ml) in HBSS.
   7. Vortex the tube on vortexer for 1 min at a speed level around 4 to 6 (if the liquid does not get up to the lid, then the speed is fine).
   8. Incubate the tube in 37 °C water bath for 3-5 min.
   9. Vortex the tube for 1 min.
   10. Sediment for 1 min by gravity until the tissue pieces settle down, collect the liquid into a new tube containing 5 ml cold FB medium.
   11. Repeat steps 2.2.6-2.2.10 3-5 times.
   12. Combine all the collections by filtering through 40 µm cell strainer to make single cell suspension.
   13. Centrifuge at 200 x g for 5 min at 4 °C.
   14. Resuspend cells in 10 ml MACS buffer, and centrifuge at 200 x g for 5 min at 4 °C.
   15. Optional: If there are a lot of blood cells, resuspend cells with 1 ml RBC lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA), keep on ice for 1 min, then dilute with 10 ml MACS buffer and centrifuge at 200 x g for 5 min. Wash one more time with MACS buffer.
3. Isolation of Thy1.2+ fibroblasts by MACS (magnetic-activated cell sorting)
   1. Determine viable cell number through trypan blue staining.
      1. Take 10 µl cells out from 10 ml cell suspension in step 2.2.14. Mix it with 10 µl 0.4% trypan blue solution.
      2. Add the mixture to a hemocytometer. Allow it to stand for 3-5 min and then examine immediately under a microscope. The dead cells are stained in blue and viable cells are unstained.
      3. Count all the viable cells in four 1 mm corner squares of the hemocytometer. Determine the viable cell number: the average count per square x 2 (dilution factor) x 10⁴ x 10 (total volume of cell suspension).
   2. For less than 1 x 10⁷ cells, resuspend cells with 10 µl Thy1.2 microbeads in 90 µl chilled MACS buffer. Add more beads proportionally if there are more than 1 x 10⁷ cells. Mix well and incubate in a refrigerator (2-8 °C) for 30 min.
   3. Add 10 ml MACS buffer and spin samples at 200 x g for 5 min; wash once with 10 ml MACS buffer and centrifuge again.
   4. Bring volume to 2 ml in MACS buffer.
   5. Set up a MACS Separator in hood. Insert an LS column to the separator. Apply 3 ml MACS buffer to the column to equilibrate it.
   6. Pass the cell suspension through the equilibrated LS column.
   7. Wash 3 times with 2 ml MACS buffer each time.
   8. Take the LS column off the Separator and elute 3 times with 2 ml MACS buffer into a new 50 ml tube.
   9. Spin at 200 x g for 5 min.
   10. Resuspend cells in 10 ml FB media, count the cells and seed into plates at proper density. For fibroblasts generated from explant culture method, the seeding cells density could be around 2-3 x 10⁴ cells/well of 24 well plate. For fibroblasts generated from enzyme digestion method, the cells density could be around 4-5 x 10⁴ cells/well of 24 well plate.

3. Generation of Retrovirus for iCM Reprogramming

Note: Perform the following steps in a BSL2 Biological Safety Cabinet under sterile conditions. The proper disposal of transfected cells, pipette tips and tubes is recommended to avoid risk of environmental and health hazards.

1. Maintain Plat-E cells in Plat-E media supplemented with 1 µg/ml puromycin and 10 µg/ml blasticidin at 37 °C with 5% CO₂.
2. On day 2, replace the medium by Plat-E culture medium lacking puromycin and blasticidin.
3. On day 0, split Plat-E cells into 10 cm dishes the day before transfection at approximately 4-5 x 10⁶ cells per plate. Cells should be ~80-90% confluent on the day of transfection.
4. On day 1, change culture media to transfection media on cells within 1 hr prior to transfection. Transfection procedure is as follows:
   1. Transfection using commercial kits
      1. Mix 20 µl transfection reagent (e.g. Lipofectamine) and 500 µl reduced serum media (e.g., Opti-MEM). Add 10 µg of the retroviral plasmid to another 500 µl reduced serum media. Incubate each mixture at room temperature (RT) for 5 min.
      2. Slowly add the plasmid mixture to transfection mixture. This step may take 15-30 sec. Incubate the mixture for 20 min at RT (solution may appear cloudy).
      3. Add mixture drop wise to the cells.
      4. Incubate overnight in a 37 °C incubator.
   2. Transfection using Calcium Phosphate.
      1. Mix 40 µl 2.5 M CaCl₂ and 440 µL ddH₂O, then add 20 µg of the retroviral plasmid (adjust the amount of ddH₂O so that total volume is 500 µl).
      2. Add 500 µl 2x HBS to a 15 ml conical tube.
      3. Vortex the HBS and meanwhile add the Calcium-DNA mixture to the HBS drop wise. This step takes around 30 sec for each sample. So maximally do 3-4 samples simultaneously.
      4. Incubate at RT for 3 min Incubation for longer time leads to larger precipitate and less cells will take up the precipitate.
      5. Add the mixture drop wise to the cells and observe under 20X microscope. Fine precipitates (a lot of small ones are good but a few large ones are not good) should be seen.
      6. Incubate overnight at 37 °C incubator.
5. On day 2, change media on cells with 8 ml prewarmed culture media. Incubate for 24 hr.
6. On day 3 and day 4, collect culture supernatant from the dishes by using a 10 ml sterile disposable syringe, filtering it through a 0.45 µm pore size cellulose acetate filter, and transferring into a 50 ml tube. Add 2 ml of retrovirus precipitation solution to every 8 ml virus-containing supernatant. Gently mix it and incubate overnight at 4 °C.
7. On day 5, spin the viral mixture at 1,500 × g at 4 °C for 30 min. The viral particles should appear in white pellet at the bottom of the tube.
8. Discard the supernatant. Spin down residual solution by centrifugation at 1,500 × g for 5 min. Remove all traces of fluid by aspiration, taking great care not to disturb the precipitated retroviral particles in pellet.
9. Resuspend retroviral pellets with 100 µl DPBS buffer for every 8 ml viral supernatant. Aliquot the virus on ice. Use immediately or store at -80 °C .

4. Reprogramming of Cardiac Fibroblasts

1. Add 4 µl of 10 mg/ml polybrene solution into the 10 ml iCM medium, and mix gently by pipetting up and down. The final concentration of polybrene is 4 µg/ml.
2. Add 500 µl iCM media containing polybrene to each well of 24 well plate seeded with fibroblasts.
3. Add 10 µl retrovirus to each well containing either 2-3 x 10⁴ cells from explant culture or 4-5 x 10⁴ cells from enzyme digestion method. The amount of virus should be proportionally increased with increased cell number in different culture plates or dishes. Put the plate in a 37 °C incubator for 24-48 hr to allow the viral infection. The time line for cardiac reprogramming is shown in Figure 1.
4. After viral transduction, replace virus containing media with 500 µl regular iCM media.
5. Change media every 2-3 days. For positive selection of viral transduced cells, add iCM media supplemented with puromycin at 2 µg/ml to target cells. Keep it for three days. After that, maintain puromycin in the iCM medium at the concentration of 1 µg/ml.
6. Three days after viral infection, take the plate out from the incubator and place it under an inverted fluorescent microscope (20X) to observe GFP expression.
   Note: Ten days after viral infection, cells could be harvested for FACS analysis and ICC analysis to determine the reprogramming efficiency (in step 5 and 6).
7. Fourteen days after viral infection, replace iCM media with B27 media. Change media every three days. Spontaneous beating cell loci may appear from three (cells from enzyme digestion method) or four (cells from explant culture method) weeks after viral transduction.

5. Immunocytochemical Analysis of Reprogramming Efficiency

1. Rinse cells with ice cold PBS three times; remove excess solution.
2. Add 0.5 ml ICC fixation buffer to each well of 24 well plates. Fix the cells at RT for 15-20 min.
3. Rinse cells with PBS three times.
4. Add 0.5 ml ICC permeabilization buffer to each well of the 24 well plates. Permeabilize the cells at RT for 15 min.
5. Rinse cells with PBS three times.
6. Add 0.5 ml ICC blocking buffer to each well of 24 well plate, incubate at RT for 1 hr.
7. Diluted primary antibodies with ICC staining buffer. Add 200 µl antibody solutions to each well and incubate overnight at 4 °C. The antibody dilution factors are listed in Materials.
8. On the next day, wash cells with PBS three times.
9. Add 200 µl secondary antibody solutions to the cells and incubate for 1 hr at RT in dark.
10. Wash cells with PBS for three times.
11. Add 150 µl mounting solution containing DAPI to each well. Allow to stain nuclei for 1 min. Then cover each well with round glass cover slip and gently press the slip against the bottom surface with a forceps to remove air bubbles.
12. Aspirate the excess solution and the samples are ready for imaging. The representative images showing the reprogrammed cells that express cardiac marker cTnT and αActinin at d14 are shown in Figure 2B.

6. FACS Analysis of Reprogramming Efficiency

1. Thoroughly wash cells once with DPBS. Add 0.3 ml trypsin (0.05%) to each well and incubate at 37 °C for 5 min.
2. Gently tap the plate to facilitate lifting the cells. Check the cell detachment under microscope. Add 1 ml FACS buffer to each well when most cells are dissociated. Pipette up and down to further mechanically dissociate the cells.
3. Transfer cells in the 24 well plate to a 96 well deep well plate well to well. Spin at 200 x g for 5 min at 4 °C to collect the cells.
4. Wash cells once with FACS buffer.
5. Add 100 µl fixation/permeabilization solutions to resuspend cells for 20 min at 4 °C.
6. Wash cells twice with 1x wash solution, pellet, and remove supernatant.
7. Prepare primary antibody solution against GFP and cTnT in 1x wash buffer. Thoroughly resuspend each sample with 50 µl antibody solution and incubate at 4 °C for 30 min.
8. Wash cells once in 1x wash solution, pellet, and remove supernatant.
9. Add 50 µl secondary antibody solution containing Alexa Fluor 488 conjugated donkey anti-rabbit IgG and Alexa Fluor 647 conjugated donkey anti-mouse IgG to each sample. Incubate at 4 °C for 30 min in dark.
10. Wash cells once in 1x wash solution, pellet, and remove supernatant.
11. Resuspend cells in 400 µl fixation buffer (DPBS with 1% PFA). Transfer cell into FACS tube with cell strainer cap. Samples are ready for FACS detection. The representative FACS analysis of reprogramming efficiency using pMxs-puromycin-MGT construct with or without puromycin selection is shown in Figure 2A.