Endoderm & Ectodermal Protocols

Hepatic

Hepatocyte Differentiation Protocol

This protocol generates functional hepatocytes from human iPSCs through a 4-phase directed differentiation spanning 25–30 days. The key innovation is the use of BMP4 activation (not BMP inhibition) during hepatic specification, which drives foregut endoderm toward hepatic rather than pancreatic or other foregut fates. The resulting hepatocytes express ALB, CYP3A4, CYP1A2, ASGR1 and exhibit functional drug metabolism, urea synthesis, and glycogen storage suitable for compound hepatotoxicity screening and precision medicine applications.

25–30

Days Total

>70%

ALB+

Phases

CYP3A4

Active

PHASE 1 Definitive Endoderm (Days 0–5)

▼

Endoderm Induction

Seed iPSCs at 300,000 cells/cm² on Matrigel in mTeSR Plus + Y-27632. Switch to RPMI + B27 + Activin A (100 ng/mL) + CHIR99021 (3 μM) on Day 0. Feed daily. By Day 5, cells express SOX17+, FOXA2+, CXCR4+. QC: Flow SOX17/CXCR4 >90%.

Activin ACHIR99021

⏲ Days 0–5

PHASE 2 Hepatic Specification (Days 5–10)

▼

Hepatic Fate Commitment

Switch to HG medium + BMP4 (20 ng/mL) + FGF2 (10 ng/mL). Unlike pancreatic differentiation, BMP4 activation (not inhibition) drives hepatic specification. Add HGF (20 ng/mL) from Day 8. Cells express HNF4A+, AFP+ (alpha-fetoprotein), TBX3+. QC: Flow HNF4A+ >70%.

BMP4FGF2HGF

⏲ Days 5–10

PHASE 3 Hepatoblast Expansion (Days 10–18)

▼

Hepatoblast Proliferation

Culture in HG medium + HGF (20 ng/mL) + Oncostatin M (OSM, 20 ng/mL) + Dexamethasone (0.1 μM). These cytokines promote hepatic progenitor expansion. Cells express ALB+ (albumin), AAT+ (alpha-1-antitrypsin), CYP3A4 induction begins. QC: ELISA confirms albumin secretion detectable (>0.1 μg/mL/day).

HGFOSMDexamethasone

⏲ Days 10–18

PHASE 4 Hepatocyte Maturation (Days 18–30)

▼

Functional Maturation

Continue HG medium + OSM (20 ng/mL) + Dexamethasone (0.1 μM) + Compound E (Notch inhibitor, 1 μM). Cells express ALB+, CYP3A4+, CYP1A2+, ASGR1+ (hepatic receptor). Functional assays: CYP3A4 activity >20 pmol/min/mg protein, urea secretion >1 μg/mL/day, albumin secretion >1 μg/mL/day, glycogen storage (PAS+), LDL uptake/release, ICG (indocyanine green) uptake/release positive. QC: Full drug metabolism panel at Day 30.

Compound ECYP3A4 AssayELISA

⏲ Days 18–30 QC

Figure 1b

Hepatocyte Differentiation Pathway

Endoderm

Days 0–5

Key Molecules:

Activin A, CHIR

Markers:

SOX17+, FOXA2+

Hepatic Spec.

Days 5–10

Key Molecules:

BMP4, FGF2, HGF

Markers:

HNF4A+, AFP+

Expansion

Days 10–18

Key Molecules:

HGF, OSM, Dex

Markers:

ALB+, AAT+

Maturation

Days 18–30

Key Molecules:

OSM, Dex, Comp.E

Markers:

CYP3A4+, ASGR1+

Gut

Intestinal Epithelial Organoid & Monolayer Differentiation

This protocol generates functional intestinal epithelia from human iPSCs through a 4-phase differentiation combining organoid and monolayer culture systems over 28–35 days. Key to this protocol is sustained WNT activation via CHIR99021 (not retinoic acid like pancreatic) to drive hindgut/midgut posterior endoderm specification. The resulting organoids and monolayers contain all major intestinal cell types: enterocytes (VIL1+), goblet cells (MUC2+), enteroendocrine cells (CHGA+), and Paneth cells (LYZ+) with functional trans-epithelial electrical resistance (TEER) suitable for barrier function studies, drug absorption assays, and disease modeling.

28–35

Days Total

>30%

Organoid Forming Efficiency

Cell Types

>300

TEER Ω·cm²

PHASE 1 Definitive Endoderm (Days 0–3)

▼

Endoderm Induction

Seed iPSCs at 250,000 cells/cm² on Matrigel in mTeSR Plus + Y-27632. Switch to RPMI + B27 + Activin A (100 ng/mL) + CHIR99021 (3 μM) on Day 0. Feed daily. By Day 3, cells express SOX17+, FOXA2+, CXCR4+. These cells are primed for hindgut specification.

Activin ACHIR99021

⏲ Days 0–3

PHASE 2 Hindgut Specification (Days 3–7)

▼

Posterior Endoderm Patterning

Maintain CHIR99021 (3 μM, sustained WNT activation for posterior fate) + add FGF4 (500 ng/mL). Unlike pancreatic (RA + SANT1) or hepatic (BMP4) protocols, sustained WNT + FGF4 drives hindgut/midgut identity. Cells express CDX2+ (hindgut marker), KLF5+. Begin 3D spheroid formation on Day 5: transfer to ultra-low attachment plates. QC: Flow CDX2+ >80%, confirm emerging spheroid budding morphology.

CHIR99021FGF4Ultra-Low Attach

⏲ Days 3–7

PHASE 3 Organoid Formation (Days 7–21)

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3D Intestinal Organoid Culture

Transfer spheroids to Matrigel domes (20 μL Matrigel per well in 48-well plates). Culture in complete medium containing the "ENR" cocktail: EGF (100 ng/mL) + Noggin (100 ng/mL) + R-spondin 1 (500 ng/mL). These factors support intestinal stem cell expansion and organoid budding. Spheroids develop budding crypts by Day 14. Cells express LGR5+ (stem), MUC2+ (goblet), CHGA+ (enteroendocrine), LYZ+ (Paneth). QC: Organoid forming efficiency >30%, confirm crypt-villus-like budding morphology by Day 21.

Matrigel DomesEGFNogginR-spondin 1

⏲ Days 7–21

PHASE 4 Monolayer Culture & Maturation (Days 21–35+)

▼

Monolayer Differentiation & Functional Assays

Dissociate organoids with Accutase and plate at 150,000 cells/cm² onto Transwell inserts (0.4 μm pore, 24-well format) pre-coated with Matrigel + laminin + fibronectin. Culture in ENR medium (continue stem cell support) or switch to ENR + DAPT (10 μM) for terminal differentiation. Medium change every 2 days. Over 2 weeks, monolayer develops functional barrier with VIL1+ (enterocytes), MUC2+ (goblet), CHGA+ (enteroendocrine), LYZ+ (Paneth), DPP4+, OLFM4+. Functional QC: TEER measurements daily — target >300 Ω·cm² by Day 35. Permeability assays (FITC-dextran, mannitol). Brush border enzyme activity (ALP, DPP4). Drug transporter studies (P-gp, BCRP substrates). Pathogen/toxin barrier integrity testing available for research applications.

Transwell InsertsDAPTTEER ElectrodePermeability Assay

⏲ Days 21–35+ Assays

Figure 1c

Gut Epithelial Differentiation Pathway

Endoderm

Days 0–3

Key Molecules:

Activin A, CHIR

Markers:

SOX17+, FOXA2+

Hindgut Spec.

Days 3–7

Key Molecules:

CHIR (sustained), FGF4

Markers:

CDX2+, KLF5+

Organoid Form.

Days 7–21

Key Molecules:

EGF, Noggin, R-spon

Markers:

LGR5+, MUC2+, CHGA+

Monolayer

Days 21–35+

Key Molecules:

ENR ± DAPT

Markers:

VIL1+, TEER+

Endoderm & Ectodermal Differentiation Protocols

Hepatocyte Differentiation Protocol

PHASE 1 Definitive Endoderm (Days 0–5)

PHASE 2 Hepatic Specification (Days 5–10)

PHASE 3 Hepatoblast Expansion (Days 10–18)

PHASE 4 Hepatocyte Maturation (Days 18–30)

Intestinal Epithelial Organoid & Monolayer Differentiation

PHASE 1 Definitive Endoderm (Days 0–3)

PHASE 2 Hindgut Specification (Days 3–7)

PHASE 3 Organoid Formation (Days 7–21)

PHASE 4 Monolayer Culture & Maturation (Days 21–35+)

Endoderm Differentiation Pathways — Comparative Overview