A) Retrovirus production
1. Plate 1.8 × 106 Plat-GP cells on poly-L-lysine-coated 10 cm dishes and culture them in DMEM containing 8% fetal bovine serum (FBS), 2 mM L-glutamine, and penicillin/streptomycin for 3 days.
2. Dilute PEI (36 µL of 1 mg/mL), retroviral plasmid DNA (10 µg; FOXA3, HNF1A, and HNF6), and VSV-G expression plasmid pCMV-VSV-G (2 µg) in 1 mL of DMEM and incubate the mixture for 15 minutes at room temperature.
3. Add the above mixture to the plated Plat-GP cells, drop-by-drop.
4. After 6 hours of incubation at 37°C, with 5% CO2, replace the medium with fresh medium, and continue culturing.
5. Collect supernatants from the transfected cells 24 hours after medium replacement. Replace the medium with fresh medium, and continue culturing.
6. Again, collect supernatants from the transfected cells 24 hours after medium replacement. Filter all the supernatants collected through 0.2 µm cellulose acetate filters and concentrate them by centrifugation (10,000 × g for 16 hours at 4°C). Resuspend the viral pellets in Hanks’ balanced salt solution (1/140 of initial supernatant volume).
B) Generation of hiHepPCs from HUVECs
1. Culture HUVECs in HUVEC medium [1:1 mixture of Medium 200, supplemented with LSGS, and FibroLife S2 Comp kit]. Following HUVEC expansion in culture, cryopreserve them until they are required.
2. Thaw the cryopreserved HUVECs, seed them at a density of 1.5 × 104 cells/well in 12-well gelatin-coated plates, and culture them in HUVEC medium. After 6 hours, replace the medium with fresh medium.
3. Twenty-four hours after seeding (5–10% confluency), add the concentrated viral supernatants and 5 µg/ml protamine sulfate to the culture medium. After centrifuging at 800 × g for 10 minutes, incubate the plates at 37°C, with 5% CO2, for 6 hours. Then, replace the medium with fresh medium and incubate the plates at 37°C, with 5% CO2, for 18 hours.
4. Retrovirus infection should additionally be conducted 3 times for 3 days.
5. After the 4th infection, replace the medium with hepato-medium (plus), composed of a 1:1 mixture of DMEM and F-12, supplemented with 20% FibroLife S2 Comp kit, 4% FBS, 1 µg/mL insulin, 10-7 M dexamethasone, 10 mM nicotinamide, 2 mM L-glutamine, 50 µM b-mercaptoethanol, 20 ng/mL rhHGF, 1 µM A8301, 2 µM SB431542, 5 µM Y-27632, and penicillin/streptomycin.
6. After 48 hours, replate the transduced HUVECs on type I collagen-coated 6-well plates, then passage them every 7 days.
C) Generation of hiHepPCs from HPBECs
1. HPBECs are obtained from adult human peripheral blood, as described previously9,10.
2. Seed HPBECs at 3 × 104 per well of type I collagen-coated 48-well plates and culture them in HPBEC medium [EGM-2MV BulletKit (Lonza), supplemented with 10% FBS and 50% FibroLife S2 Comp kit] until they reached 20–30% confluency.
3. Perform viral infection (FOXA3, HNF1A, and HNF6, with or without L-MYC), according to the protocol for generating hiHepPCs from HUVECs.
4. Additionally, perform 7 more retrovirus infections for 7 days.
5. After the 8th infection, replace the medium with a 1:1 mixture of HPBEC medium and hepato-medium (plus).
6. After 2 days, replace the medium with hepato-medium (plus), and ensure that the medium is replaced with fresh medium every week.
7. More than 1 month after the final infection, slowly expanding epithelial cell colonies appear in culture. After these colonies appear compact, perform the passage of cells.
D) Induction of hiHepPC differentiation into hepatocytes
1. Seed hiHepPCs at 5 × 103 per well of PrimeSurface ultra-low attachment 96-well plates coated with poly 2-hydroxyethyl methacrylate, as described previously11 and culture them in a medium composed of a 1:1 mixture of DMEM and F-12, supplemented with 1 µg/mL insulin, 10 mM nicotinamide, 2 mM L-glutamine, 50 µM b-mercaptoethanol, 20 ng/mL rhHGF, 1 µM Forskolin, 1 µM A8301, 20 µM Y-27632, and penicillin/streptomycin.
2. hiHepPC aggregates are observed 1 day after 3D culture initiation. Then, replace the medium with fresh medium every 3 days.
E) Induction of hiHepPC differentiation into cholangiocytes
1. Mix hiHepPCs (1 × 105) with 50 µL of Matrigel and plate them on 24-well plates.
2. After polymerization of the Matrigel on the HOT PLATE at 37°C, add an adult human bile duct-derived progenitor cell expansion medium12 (advanced DMEM/F-12, supplemented with 2 mM GlutaMax, 10 mM HEPES, N-2 supplement, B-27 supplement, 1 mM N-acetylcysteine, penicillin/streptomycin, 50 ng/mL human recombinant EGF, 100 ng/mL human recombinant Noggin, 500 ng/mL human recombinant R-spondin1, 100 ng/mL human recombinant Wnt3a, 10 µM Forskolin, 100 ng/ml human FGF10, 10 mM Nicotinamide, 10 nM gastrin, 25 ng/ml rhHGF, and 5 µM A8301).
3. After cystic epithelial spheroids are observed in the 3D culture, collect the spheroids with the medium and Matrigel from each well of 24-well plates, transfer them to 15 ml tubes, break the spheroids into small pieces by mechanically pipetting, and wash them with the medium to remove the Matrigel. Divide the broken pieces in half, mix them with new Matrigel, and resume the culturing.