Epithelial–Mesenchymal Co-culture
Layered co-culture aggregates eHFSCs with DP spheroids in a defined cocktail (CHIR99021, LDN-193189, FGF7) that recapitulates the Wnt↑/BMP↓/FGF↗ induction window.
Co-culture aggregates are assembled by depositing 8–10×10³ eHFSCs onto a single DP spheroid in a U-well, producing a layered organoid in which epithelial cells crawl over and partially encapsulate the mesenchymal core. The aggregate is then transferred into a custom defined medium that mimics the induction signaling window.
Pathway tuning: Wnt is activated via CHIR99021 (3 µM, GSK3β inhibitor); BMP is suppressed with LDN-193189 (100 nM, ALK2/3 inhibitor); FGF7 (10 ng/mL) and Shh agonist SAG (50 nM) drive early follicle germ specification. Cocktail is applied 0–6 days, then withdrawn for FGF-driven elongation.
Aggregate quality control: at day 7 we require ≥ 60 % LEF1⁺ / SOX9⁺ co-expression in the epithelial compartment as a positive marker of follicular induction.
Normalized pathway activity bias between D0–6 induction window and D7+ maintenance phase.
| Parameter | Value | Unit | Tolerance / Note |
|---|---|---|---|
| Aggregate composition | 8–10k eHFSCs + 1 DP spheroid | ||
| Wnt activator | CHIR99021 3 µM | ||
| BMP inhibitor | LDN-193189 100 nM | ||
| FGF | FGF7 10 ng/mL | ||
| Shh agonist | SAG 50 nM | ||
| Cocktail window | 0–6 | days | |
| Day-7 QC threshold | ≥ 60 % LEF1⁺/SOX9⁺ |