Intermediate Mesoderm to Self-Organizing Nephrons
The protocol commits iPSCs to posterior primitive streak with high-dose CHIR (Wnt activation), then patterns intermediate mesoderm (IM) by FGF9 + activin A, splits IM into metanephric mesenchyme and ureteric epithelium with a brief CHIR pulse, and finally allows self-organization into nephron-like structures via 3D Matrigel droplet or air-liquid interface culture. Mature organoids contain LTL+ proximal tubules, NPHS1+ podocytes, ECAD+ distal tubules, and GATA3+ collecting-duct epithelium.
CHIR99021 8 µM for 4 days in TeSR-E6 (Wnt activation).High-dose CHIR99021 8 µM in TeSR-E6 for 4 days. Wnt activation drives posterior primitive streak (T+/MIXL1+/CDX2+).
CHIR99021 5 µM to commit metanephric mesenchyme; aggregate to 3D.Brief 1-h pulse of CHIR99021 5 µM to commit to metanephric mesenchyme (SIX2+/WT1+). Then dissociate and aggregate ~5×10⁵ cells per organoid.
Withdraw growth factors. Self-organizing nephron segments by D18–25: LTL+ proximal tubules, NPHS1+/PODXL+ podocytes, ECAD+ distal/collecting epithelium.
- iPSC seeding
Plate iPSCs at low density (10–15k cells/cm²) on Matrigel in mTeSR1 + ROCK-i.
- Posterior primitive streak (Day 0–4)
Switch to TeSR-E6 +
CHIR990218 µM for 4 days. Daily medium changes. - Intermediate mesoderm (Day 4–7)
Replace with TeSR-E6 + FGF9 (200 ng/mL) + Heparin (1 µg/mL) for 3 days.
- Metanephric induction (Day 7)
1-h pulse of
CHIR990215 µM, then dissociate to single cells and aggregate ~5×10⁵ cells per organoid in low-attachment plates. - 3D culture (Day 7–25)
Place aggregates on Transwell membranes (air-liquid interface) or embed in Matrigel droplets. Maintain in TeSR-E6 + FGF9 for 5 days, then withdraw growth factors.
- Maturation & assay
Organoids develop nephron segments by D18–25. Validate by IF (LTL, ECAD, NPHS1) and bulk/single-cell RNA-seq.