Human iPSC/ESC Cardiomyocyte Induction Differentiation Kit

Reagents and Consumables Required

DMEM/F-12 (HY-K3002), DPBS (HY-K3008), Basement Membrane Matrix IPSC-qualified (HY-K6006), CEPT Cocktail Plus (1000×) (HY-K6021), Human PSC Maintenance Medium (HY-K6501), Human PSC Embryoid Body (EB) Induction Medium (HY-K6503), and 24-well TC-treated plates.

PSC Culture

Thaw ESCs (<60 passages) or iPSCs (<50 passages) according to the cell line and passage twice to obtain P3 cells. H9 and some iPSC lines have relatively limited mesoderm differentiation capacity; H1, H7, or iPSC lines with validated mesoderm performance are preferred.

Passaging Prior to Induction

1. Initiation criteria: P3 colonies should have smooth, well-defined borders, a normal nuclear-to-cytoplasmic ratio (nuclei occupying approximately 60% of the cell area), compact cell-cell contacts, and a negative mycoplasma test.

2. Prepare 12 mL Human PSC EB Induction Medium with 12 μL CEPT Cocktail Plus (1000×) and equilibrate to room temperature.

3. Select one well of a 6-well plate, wash twice with DPBS, add 1 mL ECM Gentle Dissociation Solution, and incubate at 37°C for 6-8 min.

4. Add 1 mL HBSS or DMEM/F-12 to stop dissociation, gently triturate twice, transfer to a 15 mL tube, and centrifuge at 120 × g for 5 min.

5. Resuspend in 12 mL CEPT-containing EB Induction Medium and seed into a pre-warmed matrix-coated 24-well plate at 500 μL per well.

6. After 24 h, replace with 500 μL Human PSC Maintenance Medium per well and then change medium daily until confluence is >90%.

Lateral Plate Mesoderm Pulse Activation and Expansion

1. Start when cell confluence exceeds 90%.

2. D0: Wash twice with pre-warmed HBSS or DMEM/F-12, add 500 μL Mesoderm Induction Medium per well, and perform a 24 h pulse induction.

3. D1 (24 h): Exactly 24 h later, remove medium, wash twice, add 1 mL Mesoderm Expansion Medium per well, and continue for another 24 h. The activation/expansion phase lasts 48 h in total.

Cardiogenic Mesoderm Activation and Cardiomyocyte Progenitor Specification

1. qPCR validation: After mesoderm activation/expansion, digest cells from three wells and assess Brachyury (T) and PDGFRα using the primer pairs below:

Mesoderm induction is considered successful when Brachyury and PDGFRα mRNA, calculated by ΔΔCt and normalized to β-Actin, reach approximately 120-1500-fold above the internal reference. For qPCR, use SYBR Green chemistry with 100 ng cDNA per reaction, a 60°C annealing temperature, 40 cycles, an NTC, and melt-curve analysis.

2. Wash cells twice with pre-warmed HBSS/DMEM/F-12 after confirming high marker expression.

3. Cardiogenic mesoderm activation: Add 500 μL pre-warmed Cardiac Mesoderm Induction Medium per well. Perform complete daily medium changes, with a gentle wash before replacement, for 4 d.

4. After the 4 d induction, wash twice with pre-warmed HBSS/DMEM/F-12.

5. Cardiomyocyte progenitor specification: Add 1 mL Cardiac Progenitor Cell Differentiation Medium per well and perform complete daily medium changes for 4 d. Together with the prior stage, this forms an 8 d cardiac-lineage specification program.

6. Morphology: Typical features include dark brown/black cell clusters, black ridge-like striations, nuclear condensation, smaller nuclei, and expanded intercellular spaces.

iCM Maturation and Replating

1. Prepare 6 mL Cardiac Progenitor Cell Differentiation Medium + 6 mL Induced Cardiomyocytes (iCMs) Maturation Medium + 12 μL CEPT Cocktail Plus (1000×).

2. Wash cells twice with HBSS or DMEM/F-12 and add 500 μL/well of the mixed medium for 24 h metabolic adaptation.

3. After 24 h, replace with 1 mL/well Induced Cardiomyocytes (iCMs) Maturation Medium and continue for 3 d. Synchronized spontaneous contractions generally appear as maturation progresses.

4. Mature contracting iCMs may be replated 2-3 times. Prewarm ECM-coated confocal dishes/coverslips at 37°C for 12 h and prepare complete cardiomyocyte dissociation solution by mixing Reagent A and Reagent B at 1 : 1.

5. Wash once with DPBS, add 500 μL complete dissociation solution per well, and incubate at 37°C for 12-15 min until cell gaps enlarge and cell bodies retract.

6. Centrifuge at 150 × g for 3 min, resuspend in cardiomyocyte progenitor differentiation medium containing CEPT Cocktail Plus, and seed. Keep final seeding and medium-replacement volume at ≤500 μL per dish/well.

7. After 24 h, switch to Cardiac Progenitor Cell Differentiation Medium without CEPT and culture for another 2 d before immunofluorescence analysis such as TNNT2/NKX2-5/SMA/TBX5/COL4.