Ethical approval was obtained (ACA CELL Biotech GmbH/25b-5482.2-64-1) for performing these experiments and informed consent was signed by all donors before blood extraction in compliance with institutional guidelines.

1. Preparation of mononuclear cells (MNCs) from human peripheral blood (PB)

1. Extract 30 mL of blood from healthy donors with the help of trained medical personnel according to the standard protocol.
2. Isolate PBMNCs using density gradient media according to the protocol published by Becker-Kojić et al.¹³. Isolate, by pipetting, the interphase layer between plasma and density gradient media and use sterile phosphate buffer saline (PBS) to wash the isolated cells.
3. Use a counting chamber and count the number of cells using standard methods.

2. Dedifferentiation of MNCs upon activation with human GPI-anchored glycoprotein

1. Place 6 x 10⁶ mononuclear cells in PBS/1% bovine serum albumin (BSA) in a polystyrene tube (15 mL) and incubate with the specific antibody for 30 min at 37 °C according to Becker-Kojić et al.¹³.
2. Centrifuge the cells at 300 x g at room temperature and replace PBS/BSA with Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum (FBS).
3. Grow the cells in 15 mL polystyrene tubes in a 5% CO₂ incubator at 37 °C for 8-10 days as described in Becker-Kojić et al.¹³. On day 5 (D5), add 1-2 mL of Iscove's medium supplemented with 10% FBS to each 15 mL tube.

3. Sorting of newly generated dedifferentiated cells

1. Centrifuge cultured cell suspension at room temperature for 10 min at 300 x g and aspirate with a sterile pipette the resulting supernatant according to Becker-Kojić et al.¹³.
2. Resuspend pellet obtained by centrifugation in 90 µL of cold PBS buffer (PBS pH 7.2, 0.5 % BSA, and 2 mM EDTA) and add 10 µL of CD45 positive nano-sized magnetic beads and incubate on ice for 15 min.
3. Wash the cell suspension with 2 mL of PBS buffer, centrifuge it at 300 x g for 10 min at room temperature and add 500 µL of PBS buffer to the cells and resuspend thoroughly.
4. Pipette 500 µL of pre-cooled PBS buffer into the column to wash and place it in the magnetic field using a magnetic stand.
5. Wash the cells placed on the column, twice with 500 µL of PBS buffer and collect flow-through containing CD45 negative cells in Iscove's medium supplemented with 10% FBS.
6. Use a counting chamber to determine the number of reprogrammed cells.

4. Preparation of glass coverslips for the generation of human hepatocytes

1. Separate glass coverslips (14 mm) and incubate them in nonionic detergent for 10 min. Wash glass coverslips in deionized water till no bubbles remain and incubate them in 1M HCl for 30 min (adapted from Marchenko et al.¹⁴).
2. Wash glass coverslips with deionized water at least 3x and dry them overnight at room temperature. Autoclave dried glass coverslips in a suitable container.

5. Coating cell culture plates with biolaminin for 2D hepatic differentiation of BD-PSCs

1. Place autoclaved glass coverslips with a sterile pair of tweezers in 4 well plates and turn UV lights on for 30 min to ensure sterile conditions.
2. Thaw aliquots of biolaminin and add 120 µL of 5 µg/mL biolaminin to each glass cover slip. Leave the coated glass coverslips overnight at 4 °C.
3. Remove the excess of biolaminin and add 200 µL of hepatocyte differentiation medium as described below.

6. Preparation of hepatocyte differentiation media

1. Make 500 mL of hepatoblast knockout serum replacement (KSR)/dimethyl sulfoxide (DMSO) medium consisting of 76.4% knockout DMEM (KO-DMEM), 20% KSR, 0.5% L-glutamine, 1% non-essential amino acids (NEAA), 0.1% β-mercaptoethanol, 1% DMSO, and 1% penicillin-streptomycin (Pen/Strep).
2. Prepare 500 mL of hepatocyte maturation medium containing 1% L-glutamine, 10 µM hydrocortisone 21-hemisuccinate sodium salt (HCC), and 1% Pen/Strep.
3. Aliquot medium from the stock and add fresh hepatocyte growth factor (HGF) and oncostatin M (OSM) at final concentrations of 10 ng/mL and/or 20 ng/mL for each medium change.
   ​NOTE: Oncostatin M is a cytokine belonging to the interleukin 6 group of cytokines, important for hematopoiesis as well as for liver development.

7. Culturing hepatic cells differentiated from BD-PSCs

1. Put 3 x 10⁵ BD-PSCS cells into each well of a 4-well plate coated with biolaminin.
2. Place the 4-well plates in the incubator at 37 °C and 5% CO₂. Culture the cells for 5 days in KSR/DMSO hepatoblast medium that supports endodermal differentiation and change the medium every second day.
3. Switch to hepatocyte maturation medium on day 5 and culture the cells for an additional 7-10 days in the incubator at 37 °C and 5% CO₂. Change the medium every 48 h.

8. 3D spheroid hepatic differentiation

1. Count cells using a counting chamber.
2. Centrifuge BD-dedifferentiated cell suspension at 300 x g for 10 min at room temperature. Remove supernatant and resuspend BD-dedifferentiated cells in KSR/DMSO medium at 2 x 10⁶ cells/mL.
3. Pass the cells through a 40 µm cell strainer to ensure a single-cell suspension and to remove any additional debris.
4. Count cells using a counting chamber and prepare a sufficient volume for each cell seeding density in order to dispense the required volume per well. Prepare a gradient with top seeding of 1 x 10⁶ cells to a low seeding density of 4000 cells.
5. Dispense 100 µL of KSR/DMSO medium in 96 well-low attachment plates and add 100 µL of cell seeding dilution.
6. Place low attachment plate in the incubator at 37 °C and 5% CO₂ and culture them for 5 days.
7. Change 50% of the medium from days 3-4 after seeding when the spheroids are sufficiently compact.
8. On day 5, change the medium to hepatocyte maturation medium and culture the cells for an additional 7-10 days for further maturation. Change the medium every second day.

9. Immunofluorescence analysis of newly generated 2D liver cell cultures

1. Culture the cells according to the differentiation method described above for 4, 8, 15, and 24 days and remove media.
2. Incubate cells with pre-warmed fixative consisting of 4% paraformaldehyde in PBS for 10 min. Discard the fixative and wash the cells 2x with PBS for 5 min each. Immediately add 0.1% triton X-100 solution and permeabilize the cells for 5 min. Wash 2x with PBS.
3. Add blocking solution made of PBS and 5% BSA and place on rocker plate for 1 h at room temperature.
4. Dilute primary antibodies in dilution buffer 1% BSA/PBS as follows: albumin (ALB) 1:50, alpha-1 fetoprotein (AFP) 1:250, cytokeratin 18 (CK18) 1:50, hepatocyte nuclear factor 4 alpha (HNF4α) 1:1000 and transthyretin (TTR) 1:50. Use 50 µL of antibody dilution per well.
5. Incubate the cells for 1 h at room temperature. Then discard the antibody solution, wash the cells for 5 min, and repeat the wash step 3x.
6. Prepare the following secondary antibodies in dilution buffer: rabbit anti-chicken IgG (Texas red) 1:1000, goat anti-mouse IgG (488) 1:1000, and goat anti-rabbit (488) 1:500. Use 50 µL of antibody dilution per well and incubate the cells for 30 min at room temperature.
7. Wash the cells 3x with PBS for 5 min each and mount the coverslips with mounting media containing DAPI for microscopic analysis.

10. Live staining of newly formed liver spheroids

1. Carefully discard culture media while not touching the spheroids, add freshly made PBS with 0.1% triton X-100 solution, and incubate for 5 min to permeabilize the cells.
2. Wash the spheroids with the medium by slowly pipetting for 5 min, repeat 2x.
3. Incubate the spheroids with the primary antibodies ALB (1:50), AFP (1:250), CK18 (1:50), CYP2E1 (1:200), and CYP3A4 (1:200) prepared in PBS with 1% BSA for 1 h in 5% CO₂ incubator at 37 °C. Use 50 µL of antibody dilution per well.
4. Carefully remove the excess antibody solution and wash the spheroids with medium 3x.
5. Prepare the corresponding secondary antibodies goat anti-mouse IgG (Cy3), goat anti-mouse IgG (488), and rabbit anti-chicken IgG (Texas red) at a dilution of 1:1000 and 1:500 for goat anti-rabbit (488) in PBS in 1% BSA. Use 50 µL of antibody dilution per well and incubate for 20 min in the incubator at 37 °C and 5% CO₂.
6. Carefully wash 3x with medium and leave the plate for 30 min in the incubator at 37 °C and 5% CO₂ before performing fluorescence microscopy.

11. Examination of spheroids using a fluorescence microscope

1. Switch on the fluorescence light source 10 min before use, turn on the computer and open the imaging software.
2. Use an objective with 4x magnification, click on button 4x in the toolbar to have the correct scale bar selected, then place the 96-well plate on the stage center plate.
3. Turn the LED light source on, use the brightfield filter, and position the plate to the well of interest using the x-y-axis stage adjustment knob.
4. Change to camera light path, click the button Live in imaging software to visualize the image on the screen, and ensure the spheroid is centered using the x-y-axis knobs and focus using the coarse/fine focus knob.
   NOTE: The shape of the spheroids remains constant after applying live staining.
5. Choose the Brightfield Observation method in the toolbar, put exposure settings to automatic, and click the button Snapshot in the camera control panel to take a picture. Then save the picture as .vsi file using the appropriate name in the folder of interest.
6. Place ambient light shielding plate to turn off LED light, change to filter for B-excitation, choose 488 Observation method in the toolbar, open shutter, take a picture by clicking button Snapshot, close shutter, then save file as described above.
7. Repeat this with a filter for G-excitation using the appropriate observation method (Texas red or CY3). Then reiterate the whole procedure for each well of interest.
8. Return the plate to the 5% CO₂ incubator at 37 °C and culture it as described above.