NOTE: Vendor information for all reagents used in this protocol has been listed in Table 1. All media/plates should be sterile and at least at room temperature when cells are to have direct contact with them.

1. Passaging Human Pluripotent Stem Cells (hPSCs) onto Laminin 521

NOTE: The cell passaging procedure described below is based on single cells and is ideal for the derivation of a homogenous population of hepatocyte-like cells from hPSCs. Colony plating is also applicable and has been described previously²⁴.

1. Prepare laminin-coated plates as needed.
   1. Thaw the 100 µg/mL stock of recombinant laminin 521 (LN-521) at 4 °C.
   2. Dilute the thawed LN-521 in ice-cold 1x DPBS (with Ca²⁺/Mg²⁺) to make a 5 µg/mL solution.
   3. Add 1 mL of 5 µg/mL LN-521 solution to coat one well of a 6-well plate and rock to spread it evenly in the well.
   4. Incubate the plates in a 37 °C/5% CO₂ cell culture incubator for 2 – 4 h for urgent use or in a 4 °C refrigerator overnight.
   5. Store the laminin-coated plates in a 4 °C refrigerator as required. Keep the plates on a flat surface and seal them to avoid evaporation and contamination.
      NOTE: Never let the coated wells dry out; top them up with extra 1x DPBS (with Ca²⁺/Mg²⁺) if required. Use the plates within 2 weeks.
2. Allow the required number of pre-coated plates to reach room temperature before use or incubate the plates at 37 °C for 0.5 – 1 h.
3. Carefully aspirate the coating LN-521 solution without damaging the coated surface. Note: It is critical not to damage the coated surface before seeding cells on it.
4. Immediately add 1 mL of pre-warmed-mTeSR1 medium supplemented with 10 µM Rho-associated kinase (ROCK) inhibitor Y27632 to one well of a 6-well plate. Leave the plate in the cell culture incubator to receive cells.
   NOTE: Never allow the laminin-coated wells to dry.
5. Aspirate the medium from well-maintained hPSCs at about 75% to 85% confluency. Wash the cells from one well of a 6-well plate once with 1 mL of room-temperature 1x DPBS (no Ca²⁺/Mg²⁺).
6. Add 0.5 mL of 1x Accutase to the cells and incubate at 37 °C for 6 – 8 min to dissociate the cells.
   NOTE: To check whether the digestion is long enough or not, gently tap the plate and check whether the cells can detach easily. If yes, then it is time to stop the enzymatic reaction; if not, extend the digestion for an extra 1 – 2 min.
7. Terminate the dissociation by adding 2 mL of fresh mTeSR1 medium supplemented with 10 µM Y27632 to the cells. Pipette up and down using a P1000 tip several times to make a single-cell suspension.
8. Count the viable cells using a hemocytometer. Use Trypan Blue to stain and exclude dead cells¹⁴
9. Calculate the total number of cells needed. For routine hPSC passaging, seed 4 x 10⁵ to 5 x 10⁵ cells per well of a 6-well plate (i.e., 4.21 x 10⁴ to 5.26 x 10⁴ per cm²). For passaging hPSCs for hepatocyte differentiation, seed 6.5 x 10⁵ to 7.5 x 10⁵ (i.e., 6.84 x 10⁴ to 7.89 x 10⁴ per cm²) cells per well of a 6-well plate.
   NOTE: The seeding density for each cell line might need minor optimization based on the empirical density given here for hepatic differentiation.
10. Transfer the needed cell suspension into a sterile 15-mL or 50-mL centrifuge tube and centrifuge at 115 x g for 3 min at room temperature.
11. Aspirate the supernatant slowly and then resuspend the cell pellet in fresh, warm mTeSR1 medium supplemented with 10 µM Rho-associated kinase (ROCK) inhibitor Y27632, using adequate medium to make the desired cell density.
    NOTE: The use of ROCK inhibitor is highly recommended in order to enhance cell attachment and survival rate.
12. Seed the cells to the prepared plates and rock them back and forth and side to side to evenly distribute the cells.
    NOTE: It is critical to ensure that cells are distributed evenly in the wells whether the plate is for routine cell culture or hepatocyte differentiation experimentation.
13. Place the plates in the cell incubator and maintain the cells at 37 °C/5% CO₂ for 24 h to allow them to attach and recover.
14. Examine the cells the next day and withdraw ROCK inhibitor if cell-cell contact has been established. Maintain the cells in mTeSR1 medium for routine culture or switch to differentiation medium as needed.
    NOTE: If the cells were seeded with the mentioned density, the confluency should be ideal for routine maintenance or hepatocyte differentiation.

2. Differentiating hPSCs to Hepatocyte-like Cells on Recombinant Laminins

1. Prepare differentiation medium.
   1. Make human Activin A stock solution: dissolve human Activin A powder to make a 100 µg/mL stock solution in sterile 0.2% bovine serum albumin (BSA)/DPBS. Make small aliquots and store them at -20 °C. Use at 1:1,000.
   2. Make mouse Wnt 3a stock solution: dissolve mouse Wnt 3a powder to make a 10 µg/mL stock solution in sterile 0.2% BSA/DPBS. Make small aliquots and store them at -20 °C. Use at 1:200.
   3. Make human hepatocyte growth factor (HGF) stock solution: dissolve human HGF powder to make a 10 µg/mL stock solution in sterile 0.2% BSA/DPBS. Make small aliquots and store them at -20 °C. Use at 1:1,000.
   4. Make Oncostatin M (OSM) stock solution: dissolve Oncostatin M (OSM) powder to make a 20 µg/mL stock solution in sterile 0.2% BSA/DPBS. Make small aliquots and store them at -20 °C. Use at 1:1,000.
   5. Make 500 mL of endoderm-priming stock medium: 2% B27 supplement (50x, minus vitamin A) and 1% penicillin/streptomycin (final concentrations at 100 IU/mL and 100 µg/mL, respectively); top up to 500 mL using Roswell Park Memorial Institute 1640 (RPMI 1640) basal medium. NOTE: Store the stock at 4 °C and use within two weeks. Aliquot medium from the stock and add fresh Activin A and Wnt 3a (final concentrations at 100 ng/mL and 50 ng/mL, respectively) at each medium change.
   6. Make 500 mL of KSR/DMSO differentiation medium: 80% knockout DMEM (KO-DMEM), 20% knockout serum replacement (KSR), 0.5% GlutaMAX, 1% non-essential amino acids (NEAA), 0.1 mM beta-mercaptoethanol, 1% DMSO, and 1% penicillin/streptomycin (final concentrations at 100 IU/mL and 100 µg/mL, respectively). Filter under vacuum. Store at 4 °C and use within two weeks.
   7. Make 500 mL of HepatoZYME maturation medium: 1% GlutaMAX, 10 µM hydrocortisone 21-hemisuccinate sodium salt (HCC), and 1% penicillin/streptomycin (final concentrations at 100 IU/mL and 100 µg/mL, respectively); top up to 500 mL using HepatoZYME basal medium. NOTE: Store the stock at 4 °C and use within two weeks. Aliquot medium from the stock and add fresh HGF and OSM (final concentrations at 10 ng/mL and 20 ng/mL, respectively) for each medium change.
2. Seed hPSCs for hepatocyte differentiation on LN-521, as described in section 1. If LN-521/LN-111 is to be used as the substrate, coat the plates with LN-521 and LN-111 (1:3 ratio) at the final laminin concentration of 5 µg/mL; the rest treatment should be the same as pure LN-521-coated plates.
   NOTE: LN-521/LN-111 is not ideal for routine culture of hPSCs; it is only used for differentiation experiments.
3. Check the cell confluency 24 h after seeding. Initiate cellular differentiation once the cell confluency reaches about 40%. Remove the spent mTeSR1 medium and add fresh endoderm-priming medium supplemented with 100 ng/mL Activin A and 50 ng/mL Wnt 3a. Call this differentiation day 1.
   NOTE: It is highly recommended to initiate the differentiation the day after seeding the cells.
4. Change the endoderm-priming medium every 24 h for 3 days for human embryonic stem cells (hESCs). As for human induced pluripotent stem cells (hiPSCs), extend this stage for 2 more days to prime the cells to definitive endoderm-like cells, but use the endoderm-priming medium supplemented only with 100 ng/mL Activin A for these two days¹².
   NOTE: To ensure successful endoderm specification, one can examine the expression of endodermal markers, such as FOXA2 and SOX17. According to immunofluorescence staining, over 80% of the derived cells are positive for both markers in our lab.
5. Switch to KSR/DMSO differentiation medium on day 4 (for hESCs)/day 6 (for hiPSCs). Change the medium daily for the first 3 days and then on the fifth day of this differentiation stage.
   NOTE: No feeding is needed on the fourth day of this differentiation stage. Use unsupplemented KO-DMEM to wash the cells once before medium change if there are many dead cells. To check whether the differentiation for this stage is successful or not, one can test the expression of hepatic progenitor cell markers, such as AFP, CK19, and HNF4A. Nearly 90% of the cells will be positive for these markers based on our experience.
6. After 5 days of the KSR/DMSO differentiation stage, switch to the HepatoZYME maturation stage. Wash the cells once with plain HepatoZYME basal medium after removing the KSR/DMSO medium. Add HepatoZYME maturation medium supplemented with 10 ng/mL HGF and 20 ng/mL OSM.
7. Change the medium every 48 h for 7 – 10 days, at which point the cells are ready for standard characterization or further use.
   NOTE: Hepatocyte marker expression examinations, metabolic function tests (such as cytochrome P450 activity), urea and albumin secretion tests, glycogen storage tests, and Indocyanine green (ICG) uptake tests are typical characterization methods.
   NOTE: The timeline of the differentiation protocol is shown in Figure 5. In our lab, we routinely check the derived HLCs' hepatocyte-specific marker expression levels, albumin secretion, and cytochrome P450 (CYP) 3A and 1A2 activity.