iPSC-Derived Pancreatic β-Cells & SC-Islets

Six-Stage SC-β Differentiation

I use a refined Pagliuca/Rezania-style protocol, taking iPSCs through definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitors, endocrine progenitors, and finally insulin-producing β-like cells. Each transition is gated by signaling cocktails (Activin A, KGF, retinoic acid, γ-secretase inhibition, T3, AXL inhibition) and validated by lineage-defining transcription factors.

1. Stage 1 — Definitive Endoderm

   Plate iPSCs at high density; treat with Activin A + CHIR99021 (Day 1 only) for 3 days. Validate >80% SOX17+/FOXA2+ by flow.

2. Stage 2–3 — Gut tube and posterior foregut

   Switch to KGF, then add retinoic acid + Cyclopamine + LDN-193189 to commit to PDX1+ pancreatic identity.

3. Stage 4 — Pancreatic progenitors

   Continue KGF + EGF; sort or quantify PDX1+/NKX6.1+ co-positivity; expect >50% efficiency.

4. Stage 5 — Endocrine commitment

   Add γ-secretase inhibitor XXI to silence Notch and induce NEUROG3+ endocrine progenitors. Co-treat with T3, SB431542 and heparin.

5. Stage 6 — Aggregation & maturation

   Aggregate cells in low-attachment plates or rotational culture (~75 µm clusters). Mature 7–14+ days in CMRL + ZnSO₄ + AXL inhibition. Validate by static GSIS and dynamic perifusion.

Glucose-Stimulated Insulin Secretion

Mature SC-islets are starved in 2.8 mM glucose, then sequentially challenged with 2.8 mM, 16.7 mM, and 16.7 mM + 30 mM KCl. Static GSIS quantifies stimulation index (SI = high/low ratio >2 considered functional). Dynamic perifusion captures first- and second-phase insulin release with <1-min temporal resolution.

Multi-Modal Validation

Validation combines C-peptide/insulin co-staining (with C-peptide as the bona fide marker of de novo synthesis), DTZ live-cluster staining for zinc-rich granules, electron microscopy for mature secretory granules, and bulk + single-cell RNA-seq for α/β/δ cell identity assignment. See the Genomics & NGS page for representative pipelines.

Type 1, Type 2, MODY & Stress Models

SC-islets are stressed with cytokine cocktails (IFNγ + IL-1β + TNFα) for type-1-like immune attack; with palmitate or high glucose for type-2-like glucolipotoxicity; or compared genotype-matched to MODY-mutant lines (HNF1A, GCK, HNF4A) for monogenic diabetes. ER-stress (thapsigargin) and oxidative-stress (H₂O₂) challenges complete the panel.