Wnt-Modulated (GiWi) Cardiac Differentiation
Sequential Wnt activation (Day 0–2 with CHIR99021) followed by Wnt inhibition (Day 3–5 with IWP-2 or Wnt-C59) drives high-efficiency cardiomyogenesis through mesoderm. Spontaneous beating typically appears by Day 8–10. Lactate-medium selection from Day 10–14 enriches cardiomyocytes by exploiting their unique ability to oxidize lactate for ATP production.
Single-cell dissociate; plate at ~3×10⁵/cm² on Matrigel in mTeSR1 + Y-27632 10 µM.
CHIR99021 6 µM (24 h pulse).IWP-2 5 µM or Wnt-C59 2 µM.RPMI + B27 minus insulin + CHIR99021 6 µM (24 h pulse). CHIR concentration is line-dependent (4–9 µM); titrate per line.
Glucose-free RPMI + 4 mM sodium lactate × 4–6 days. Non-CMs die because they cannot oxidize lactate. Validate >90% cTnT+ by flow.
Replate on PDMS or fibronectin in maturation medium with T3, IGF-1, fatty acids, dexamethasone. Apply electrical pacing (1–2 Hz) or stiffness substrate. Adult-like sarcomeres + KCNJ2/SERCA2a maturation.
- iPSC plating
Single-cell dissociate; plate at ~3×10⁵ cells/cm² on Matrigel in mTeSR1 + 10 µM Y-27632.
- Wnt activation (Day 0)
Switch to RPMI + B27 minus insulin with
CHIR990216 µM for exactly 24 h. CHIR concentration is line-dependent (4–9 µM). - Wnt inhibition (Day 3)
Add
IWP-25 µM orWnt-C592 µM; refresh medium daily. Discontinue at Day 5. - Cardiac induction (Day 7–10)
Switch to RPMI + B27 (full); spontaneous contractions appear in 2–3 days.
- Lactate selection (Day 10–14)
Switch to glucose-free RPMI + 4 mM sodium lactate for 4–6 days; non-CMs die. Verify >90% cTnT+ by flow.
- Maturation (Day 30–60)
Replate on PDMS or fibronectin; mature in maturation medium (T3, IGF-1, dexamethasone, fatty acids); apply electrical pacing or stiffness cues.