Human iPSC/ESC Hepatocyte Induction Differentiation Kit

Reagents and Consumables Required

DMEM/F-12 (HY-K3002), DPBS (HY-K3008), Basement Membrane Matrix IPSC-qualified (HY-K6006), iPSC/ESC Dissociation Solution (Enzyme-Free) (HY-K6020), CEPT Cocktail Plus (1000×) (HY-K6021), ECM Gentle Dissociation Solution (HY-K6022), Human PSC Maintenance Medium (HY-K6501), Human PSC Embryoid Body (EB) Induction Medium (HY-K6503), and low-attachment 6/96-well plates.

Thaw the kit overnight at 4°C before use and use promptly after thawing. Aliquot if necessary to minimize repeated freeze-thaw cycles.

Adherent Cell-Initiated Differentiation Protocol

PSC Culture

Thaw ESCs (<60 passages) or iPSCs (<50 passages) and passage twice to obtain P3 cells. Restart from a new thaw if colonies show abnormal morphology, spontaneous differentiation, or excessive detachment.

Definitive Endoderm Induction

1. Start when P3 cells form small island-like colonies at approximately 80% confluence without single-cell stress or stress-induced spheroids.

2. Remove PSC maintenance medium, wash twice with DMEM/F-12, and add Definitive Endoderm Induction Medium at 2 mL/well for 6-well plates, 1 mL/well for 12-well plates, or 0.5 mL/well for 24-well plates. Replace medium daily until 100% confluence; this stage generally lasts 3-4 d.

Hepatic-Specified Endoderm Induction

Add Hepatic Specification Endoderm Induction Medium at 2 mL/well (6-well), 1 mL/well (12-well), or 0.5 mL/well (24-well). Replace medium daily for 4 d.

Hepatic Progenitor Induction

Add Hepatic Progenitor Cell Induction Medium at the same plate-format volumes, replace medium every 2 d, and continue for 6 d.

Hepatocyte Induction

Add Hepatocyte Induction Medium at the same plate-format volumes, replace medium every 2-3 d, and continue for 8 d. The resulting hepatocyte-like cells can then be used for phenotypic and functional assays.

Reference Procedure for Suspension or Matrix-Embedded Culture

If suspension or matrix-embedded culture is needed, dissociation may be performed during the hepatic progenitor stage (approximately Day 10-13).

1. Wash twice with DPBS.

2. Add ECM Gentle Dissociation Solution and incubate at 37°C for 10-15 min.

3. Stop when cells become rounded, shrunken, and bright and a small number detach with gentle agitation. If extensive detachment occurs, add an equal volume of Hepatocyte Induction Medium.

4. Gently resuspend, transfer to a 15/50 mL tube, and centrifuge at 120 × g for 5 min. Resuspend in Hepatocyte Induction Medium using 2 mL per dissociated well (6-well format) and supplement CEPT Cocktail Plus to 1×.

5. Seed 100 μL per well into a low-attachment U-bottom 96-well plate, centrifuge at 1500 rpm for 5 min, and culture overnight at 37°C.

6. On Day 2 add 100 μL Hepatocyte Induction Medium without CEPT per well. On Day 3 completely replace with 100 μL fresh medium without CEPT.

Embryoid Body (EB)-Initiated Differentiation Protocol

1. Use healthy P3 PSCs prepared as described above.

2. Initiate EB induction at approximately 80% confluence. Wash twice with DPBS, add HY-K6020 or HY-K6022, and incubate at 37°C for 6-10 min. Stop dissociation when cells become rounded, shrunken, bright, and begin to detach.

3. Collect cells and centrifuge at 120 × g for 5 min. When HY-K6020 is used, resuspend in EB Induction Medium at 500 μL per original well; when HY-K6022 is used, use 4 mL per original well. Supplement CEPT Cocktail Plus to 1×.

4. Seed 100 μL per well into low-attachment U-bottom 96-well plates, centrifuge at 1500 rpm for 5 min, and incubate overnight. On Day 2 add 100 μL EB Induction Medium without CEPT.

5. Definitive endoderm: On Day 3 replace with 200 μL Definitive Endoderm Induction Medium per well and perform daily half-medium changes for 3 d.

6. Hepatic specification: Add 200 μL Hepatic Specification Endoderm Induction Medium per well and perform daily half-medium changes for 4 d.

7. Hepatic progenitor: Add 200 μL Hepatic Progenitor Cell Induction Medium per well and completely replace medium every 2 d for 6 d.

8. Hepatocyte induction: Add 200 μL Hepatocyte Induction Medium per well, perform half-medium changes every 2-3 d, and continue for 8 d.