Paraformaldehyde (pfa)

To quantify the protein expression level of HLCs, hHF-iPSCs were used as the control group, and ALB, AFP, CYP3A4, and CYP7A1 were selected as hepatocyte marker proteins, which were identified by immunofluorescence staining. hHF-iPSCs, HLCs, and frozen sections of hepatic tissue were fixed with 4% paraformaldehyde (Biolegend) for 20 min and then permeabilized by 0.1% Triton-X in PBS for 10 min. Cells were then blocked with 5% BSA for 1 h and stained with primary antibodies (SOX17, ALB, AFP, CYP3A4, and CYP7A1) at 4 °C overnight. All of the primary antibodies diluted at a ratio of 1:100 were from Santa Cruz Biotechnology. The primary antibody which was used to detect the formation of DE was mouse anti-SOX17 antibody. Additionally, primary antibodies including mouse anti-ALB, rabbit anti-AFP, rabbit anti-CYP3A4, and mouse anti-CYP7A1 were selected to detect the corresponding genes. Cells were washed three times with PBS before incubation with secondary antibodies: Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500; Invitrogen) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:500; Invitrogen). Nuclei were stained with DAPI (US Everbright Inc.) at 25 °C for 2 min. Images were captured with an Olympus IX71 fluorescence microscope. We used ImageJ to quantify the percentage of positive SOX17, ALB, AFP, CYP3A4, and CYP7A1 populations from three pictures.

The BM samples were incubated with lysis buffer (for 10 min-1 h) (Biolegend) at room temperature. Afterwards, cells were washed twice, first with lysis buffer and secondly with PBS-BSA 0.5% (Sigma-Aldrich) and stained with anti-human CD45-PerCP, anti-human CD117-APC, anti-human CD203c-PECy7, anti-human FcεRI-PE, anti-human MRGPRX2-PE, anti-human CD300a-PE, anti-human CD63-PE and anti-human CD25-FITC. Cells were stained for 15 min at 4°C in the dark and washed with PBS-BSA 0.5% and suspended in PBS supplemented with 0.1% paraformaldehyde (Biolegend).

Macrophages were mechanically harvested after 7 to 12 days of differentiation/treatment using a cell scraper (Greiner Bio-one, Kremsmünster, AT), spun at 1000 rpm/10 min, and analyzed for cell integrity and viability by microscopy using Trypan blue exclusion or a Live/Dead cell viability dye (Ghost Dye^TM Violet 510, Tonbo Biosciences, San Diego, CA, USA). For immunophenotyping, viable cells were stained using specific fluorophore-conjugated anti-human monoclonal antibodies (Table 1) at concentrations recommended by the manufacturer. Cells were incubated with Abs for 30 min at +4 °C in the dark, washed using PBS/0.1% BSA (FACS Wash) buffer, and fixed in 4% Paraformaldehyde (Biolegend, San Diego, CA, USA) before analyzing at BD FACS Verse^TM flow-cytometer (BD Biosciences, San Diego, CA, USA). Live/dead dye was routinely applied to exclude dead cells before analyzing cells of interest. Data were analyzed using FACS Suite or Diva software v 9.0 (BD Biosciences, San Diego, CA, USA).