Jun 02, 2025

Public workspace[MDC_Alternative_2D_hiPSC-CM_diff] hiPSC Cardiomyocyte differentiation V.1

DOI
https://dx.doi.org/10.17504/protocols.io.14egn9p8yl5d/v1
  • Yuichiro Ueda1,
  • Carolin Genehr1
  • 1Max Delbrück Center for Molecular Medicine
Yuichiro Ueda
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DOI: https://dx.doi.org/10.17504/protocols.io.14egn9p8yl5d/v1
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 02, 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.
Troubleshooting
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 Concentration10 micromolar (µM) ROCK inhibitor for the first Duration24:00:00 .
Incubate in a hypoxia incubator (Concentration5 % CO2 , Concentration5 % O2 , Temperature37 °C ).
Change the medium to E8 without ROCK inhibitor after Duration24:00:00 , and continue daily media changes until the cell density reachesConcentration90-95 % (approximately 3-4 days). *Never reach full confluency


hiPSC in E8 (day4) before differentiation, 90% confluency

3d
Critical
Day 0-21 Cardiac Differentiation
1w 2d
Day 0 (Priming): Change the medium to Amount2 mL /well of Cardiac: priming medium (RPMI 1640 + B27 Minus Insulin + CHIR-99021). Use a CHIR concentration between Concentration5-10 micromolar (µM) depending on the individual hiPSC line.
Day 1: Add Amount4 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 Amount4 mL of Cardiac: induction medium/well (RPMI 1640 + B27 Minus Insulin + IWR-1-endo). Use Concentration5 micromolar (µM) IWR-1-endo for our iPSC.
2d
Day 5: Add Amount4 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 Amount2-3 mL Maintenance medium (RPMI 1640 + B27)

Day 8: Remove Maintenance medium and replace with Amount2-3 mL Maintenance medium (RPMI 1640 + B27) Beating cells should be observed around days 7-10.
Days 9-14: Change Maintenance medium Amount3 mL every Duration48:00:00 .

2d
Days 9-12 (Optional Lactate Selection): If desired, perform metabolic selection. Replace Maintenance medium with Amount2 mL Lactate Selection medium/well (RPMI 1640 w/o glucose + CDM3 supplement + Sodium DL-lactate Amount50 µL ). Check for cell death and replace Lactate Selection medium as needed. After Duration24:00:00 , revert to Maintenance medium if a high proportion of beating hPSC-CMs is observed.
1d
Days 15-21: Perform media changes Amount3-5 mL every Duration24:00:00 to Duration48:00:00 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.