Directed cardiomyocte generation from human pluripotent stem cells using a chemically defined protocol (GiWi2)

Matrigel ( life science, cat# 354230 )1. Y27632 ( Tocris, cat# 1254 ), 5 mM Y27632 stock (1:1000)

1. TeSR-E8 ( Stem Cell Technologies, cat# 05990 ): Thaw TeSR-E8 25X Supplement at room temperature (15-25℃) or overnight at 2 - 8℃. Add 20 mL of TeSR-E8 25X Supplement to 480 mL of TeSR-E8 Basal Medium. Mix thoroughly. Store complete medium at 2 - 8℃ for up to 2 weeks.
2. Accutase ( Innovative Cell Technology, cat# AT104)
3. CHIR99021 ( Selleckchem, cat# S1263-25mg ): 10 mM CHIR99021 stock
4. IWP2 ( Tocris, cat# 3533-10mg ): 5 mM IWP2 stock
5. Insulin ( Sigma, cat# 91077C-250MG ): 5 mg/mL in stock (1:500)
6. RPMI ( Life Technologies, cat# 11875-085 )
7. B-27 Minus Insulin (Gibco, cat# A18956-01 ), 50X for RPMI-B27 [RPMI1640 (Life Technologies); 2% B-27 Supplement Minus Insulin (B27-Insulin)]
8. L-Ascorbic acid 2-phosphate sesquimagnesium ( Sigma, cat# A8960-5G ): 50 mg/mL in stock (1:250)
9. Cardiomyocyte Maintenance Medium (CMM) : RPMI1640 supplemented with 10 μg/mL of insulin and 200 μg/mL of L-Ascorbic acid 2-phosphate sesquimagnesium. Add 0.5 mL of insulin stock (5 mg/mL) and 1 mL of ascorbic acid (50 mg/mL) to 250 mL of RPMI1640 basal medium. Sterilize by filtration and store at 4°C.
10. Freezing medium (2X) : 20% DMSO, 60% defined FBS, 20% hES cell culture medium, and need to add fresh 10 μM Y27632 (ROCKi).

Prepare a Matrigel-coated 6-well plate and store it at 4°C, put it at room temperature at least 1 hour before use.

Remove a frozen cell vial of hPSCs, from a liquid N₂ tank and immerse the vial in a 37°C water bath without submerging the cap. Swirl the vial gently for 2 - 3 min until the contents are almost completely thawed.  

Spray the vial with 70% (v/v) ethanol and place it in a tissue culture hood. Use a sterile 1-mL pipette to gently transfer the cells into a sterile 15-mL conical tube containing 5 mL of room temperature mTeSR1 medium. Rinse the cell tube a few times without disturbing "little" cell clusters.

Centrifuge the cells at 200 g for 5 min at room temperature. Aspirate and discard the supernatant with a sterilized electronic pipette. Resuspend the cell pellet in 2 mL of TeSR-E8 medium(with 5 μM ROCKi), and gently resuspend the cells without disturbing "little" cell clusters.

Aspirate the liquid from the wells of the Matrigel-coated plate.

Slowly add the cell suspension to the Matrigel-coated 6-well plate. Put the plate back into the 37°C, 5% CO₂incubator.

The next day, aspirate the medium in each well and replace it with 2 mL of fresh room temperature mTeSR-E8 medium (without ROCKi). Repeat this medium replacement daily until cells are ready for passage.

Passage the cells with EDTA-method: Asprite the medium and wash once with DPBS. Add 1 mL 0.5 mM EDTA-DPBS buffer to each well to dissociate the cells. Incubate for 3 - 5 min at 37°C and observe with microscopy. Asprite the EDTA buffer. Add 1 mL TESR-E8 with Rock-inhibitor (1:1000) to the cells and gently detach them by pipetting them up and down. Transfer the cells to a 15 mL tube with 5 mL TESR-E8 (with ROCKi), and rinse the well with 1 mL medium. Add the cells to a Matrigel-coated plate (6 wells).

(Day -2) Passaging hPSCs using Accutane:

Take hPSCs cultured on Matrigel-coated 6-wells in mTeSR1 medium at 80-90% confluence. Aspirate medium and wash once with DPBS. Add 1 mL of room-temp Accutase to each 6-well. Place plate in 37°C, 5% CO₂ incubator for ~5 min.

Add 0.5 mL mTeSR1 to each well and gently detach the cells by pipetting the cells up and down and pool the cells in a 15-mL conical tube. Count total cells and centrifuge at 200 g for 5 minutes at room temp.

Aspirate supernatant, resuspend cells in TeSR-E8 + 5 µM Y27632, and plate 200 k cells/cm² (i.e. ~700 k cells/3.5 cm² for 12-well) onto Matrigel-coated plate. Add TeSR-E8 + 5 µM Y27632 medium to each well to obtain a final volume of 1 mL in each well of the 12-well plate. Incubate the cells. Move the plate in quick, short, back-and-forth, and side-to-side motions to disperse the cells across the surface of the wells.

Day -1 Maintenance: On day -1 of differentiation, aspirate the medium and replace it with 2 mL of room temperature TeSR-E8 per well of the 12-well plate.

Day 0 Add GSK3 inhibitor CHIR99021: On day 0 of differentiation, prepare 2 mL of 6 µM CHIR99021 in RPMI-B27 medium for each well of 12-well. Aspirate the old medium and add 2 mL of this mixture to each

well. Record the time, as the medium needs to be changed exactly 24 hours later. .

Day 1 Remove GSK3 inhibitor CHIR99021. After 24 h, aspirate medium from each well and replace with 2 mL of room-temp RPMI-B27. Put the plate back into 37°C, 5% CO₂ incubator.

Day 3 add Wnt inhibitor IWP2: On day 3 of differentiation, prepare the combined medium by using a 5 mL pipette to collect 1 mL of medium from the 12-well plate; mix it with 1mL of fresh RPMI-B27 medium in a 15 mL conical. Add 1 μl of 5 mM IWP2 (final conc. is 2.5 µM) into the 2 mL of combined medium. Before aspirating 1 mL of the remaining medium in the well of the 12-well plate, gently rock the plate back and forth to get cell debris into suspension, ensuring that the cell debris will be discarded via aspiration. Aspirate the remaining 1 mL of medium from each well of the 12-well plate, and then add 2 mL per well of the combined medium containing IWP2 to each well and incubate the cells.

Day 5 Remove Wnt inhibitor IWP2: On day 5 of differentiation, aspirate the medium from each well of the 12-well plate and add room-temperature RPMI-B27 medium at a volume of 2 mL per well. Put the plate back into the 37°C, 5% CO₂ incubator.

Day 7 Change medium with CMM: On day 7 of differentiation and every 2 days thereafter, aspirate the medium from each well of the 12-well plate and add room-temperature RPMI medium supplemented/filtered together with B27(with insulin) and 10 µg/mL insulin and 200 µg/mL L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate at a volume of 2 mL per well. Put the plate back into the 37 C, 5% CO₂ incubator.

Robust spontaneous contraction should occur by day 12. The cells can be maintained with this spontaneously beating phenotype for more than 6 months.

1. Lian X, Zhang J, Azarin SM, Zhu K, Hazeltine LB, Bao X, Hsiao C, Kamp TJ, Palecek SP. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nat Protoc. 2013 Jan;8(1):162-75. doi: 10.1038/nprot.2012.150. Epub 2012 Dec 20. PMID: 23257984; PMCID: PMC3612968.

2. Lian X, Bao X, Zilberter M, Westman M, Fisahn A, Hsiao C, Hazeltine LB, Dunn KK, Kamp TJ, Palecek SP. Chemically defined, albumin-free human cardiomyocyte generation. Nat Methods. 2015 Jul;12(7):595-6. doi: 10.1038/nmeth.3448. PMID: 26125590; PMCID: PMC4663075.