Jun 03, 2025
Version 1
[MDC_Alternative_2D_hiPSC-CM_diff] hiPSC Cardiomyocyte differentiation V.1
- Yuichiro Ueda1,
- Carolin Genehr1
- 1Max Delbrück Center for Molecular Medicine
Protocol Citation: Yuichiro Ueda, Carolin Genehr 2025. [MDC_Alternative_2D_hiPSC-CM_diff] hiPSC Cardiomyocyte differentiation. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn9p8yl5d/v1Version created by Yuichiro Ueda
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 12, 2025
Last Modified: June 03, 2025
Protocol Integer ID: 124261
Keywords: hipsc cardiomyocyte differentiation this protocol, hipsc cardiomyocyte differentiation, derived cardiomyocyte, beating cardiomyocyte, induced pluripotent stem cell, pluripotent stem cell, robust cardiac differentiation through sequential modulation, robust cardiac differentiation, wnt signaling, 2d differentiation method, mdc-alternative-2d-hipsc, differentiation
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Abstract
This protocol outlines a small molecules based, 2D differentiation method for generating high-purity human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Optimized for reproducibility across hiPSC lines, the procedure achieves robust cardiac differentiation through sequential modulation of Wnt signaling, yielding functional beating cardiomyocytes by days 7–10.
Day-3 Matrix coating of Culture Vessels
10m
Coating of culture vessels (Geltrex, Laminin-511, etc.) according to optimize protocol for hiPSC
10m
Day-3 Seeding hiPSCs
3d
Passage hiPSCs as single cells using Accutase or 1x TrypLE after 1x PBS(-) wash
Seed hiPSCs on Matrix-coated 6-well plates at a density of 4x10^5 cells/well in E8 medium supplemented with 10 micromolar (µM) ROCK inhibitor for the first 24:00:00 .
Incubate in a hypoxia incubator (5 % CO2 , 5 % O2 , 37 °C ).
Change the medium to E8 without ROCK inhibitor after 24:00:00 , and continue daily media changes until the cell density reaches90-95 % (approximately 3-4 days). *Never reach full confluency
hiPSC in E8 (day4) before differentiation, 90% confluency
3d
Day 0-21 Cardiac Differentiation
1w 2d
Day 0 (Priming): Change the medium to 2 mL /well of Cardiac: priming medium (RPMI 1640 + B27 Minus Insulin + CHIR-99021). Use a CHIR concentration between 5-10 micromolar (µM) depending on the individual hiPSC line.
Day 1: Add 4 mL basal Differentiation medium/well (B27-minus insulin, w/o CHIR-99021) without removing the previous Cardiac: priming medium.
1d
Day 3 (Induction): Remove the basal Differentiation medium and add 4 mL of Cardiac: induction medium/well (RPMI 1640 + B27 Minus Insulin + IWR-1-endo). Use 5 micromolar (µM) IWR-1-endo for our iPSC.
2d
Day 5: Add 4 mL basal Differentiation medium/well (B27-minus insulin, w/o IWR-1-endo) without removing the previous Cardiac: induction medium.
Day 7: Remove Differentiation medium and replace with 2-3 mL Maintenance medium (RPMI 1640 + B27)
Day 8: Remove Maintenance medium and replace with 2-3 mL Maintenance medium (RPMI 1640 + B27) Beating cells should be observed around days 7-10.
Days 9-14: Change Maintenance medium 3 mL every 48:00:00 .
2d
Days 9-12 (Optional Lactate Selection): If desired, perform metabolic selection. Replace Maintenance medium with 2 mL Lactate Selection medium/well (RPMI 1640 w/o glucose + CDM3 supplement + Sodium DL-lactate 50 µL ). Check for cell death and replace Lactate Selection medium as needed. After 24:00:00 , revert to Maintenance medium if a high proportion of beating hPSC-CMs is observed.
1d
Days 15-21: Perform media changes 3-5 mL every 24:00:00 to 48:00:00 with Maintenance medium depends on the color (yellowish: metabolites such as lactate) of medium.
hiPSC-CMs (d20, 1 day Lactate selection) Yield: 146.2 Mio from 18 wells of 6-well plate
3d
Notes
Ensure that all steps are performed with sterile techniques.
Optimize CHIR concentration (5-10 µM) for each cell line.
Be gentle during cell resuspension to avoid osmotic shock and shear stress.