Viruses | Free Full-Text | A Comparative Study of Human Pluripotent Stem Cell-Derived Macrophages in Modeling Viral Infections

H1 cells and induced pluripotent stem cells (iPSCs) culture, macrophage differentiation, co-culture with Huh7 cells, flow cytometry analysis, and immunofluorescence were performed as described previously [14,15]. The H1 cells (WiCell Research Institute, Madison, WI, USA) and the CD34-iPSCs [14,16] were maintained in the TeSR-E8 (STEMCELL Technologies, Vancouver, BC, Canada) medium on Matrigel (Corning, Glendale, AZ, USA)-coated plates. For the macrophage differentiation, hPSCs were dissociated into single-cell suspension with Accutase (Invitrogen, Carlsbad, CA, USA) and were seeded onto a Matrigel-coated 12-well plate at a density of 3 × 10⁴ cells per cm² in the TeSR-E8 medium with 8 μM of Y27632 (Selleck Chemicals, Houston, TX, USA). After 18–24 h, the medium was changed to an AATS medium consisting of the RPMI1640 (Gibco, Grand Island, NY, USA) medium with 500 μg/mL of recombinant human serum albumin (OsrHSA), 200 μg/mL of ascorbic acid 2-phosphate magnesium (Sigma-Aldrich, St. Louis, MO, USA), 5 μg/mL of human Apo-transferrin (Sigma-Aldrich, St. Louis, MO, USA), and 5 ng/mL of sodium selenite (Sigma-Aldrich, St. Louis, MO, USA) [17]. In the first 24 h, 5 ng/mL of recombinant human BMP-4 (PeproTech, Rocky Hill, NJ, USA) was added to induce mesoderm differentiation. Next, the medium was changed to the AATS medium supplemented with 5 ng/mL of BMP-4 and 2 μM of CHIR99021 (Selleck Chemicals, Houston, TX, USA) for another 48 h to obtain vascular mesoderm cells (VMCs). Afterwards, the VMCs were dissociated into single-cell suspension with Accutase and were seeded onto a Matrigel-coated 12-well plate at a density of 6 × 10⁴ cells per cm² in an HPC induction medium consisting of the serum-free medium (SFM) [RPMI1640 medium with 2% (v/v) of B27 Supplement (Gibco), 50 μg/mL of ascorbic acid, 1% (v/v) of GlutaMAX Supplement (Gibco, Grand Island, NY, USA), 1% (v/v) of MEM Non-Essential Amino Acids (NEAA) Solution (Gibco, Grand Island, NY, USA)], 50 ng/mL of human VEGF165 (Sino Biological, Beijing, China), and 10 ng/mL of bFGF (Sino Biological, Beijing, China). After 48 h of VEGF and bFGF induction, the medium was changed to the HPC differentiation medium supplied with 10 μM of SB431542 (Selleck Chemicals, Houston, TX, USA) to promote the formation of hemogenic endothelial cells (HECs) and the generation of HPCs. After 72 h of the SB431542 induction, the floating HPCs in the culture were collected, transferred to another new plate, and incubated for 6 days for iMAC differentiation in the macrophage induction medium consisting of SFM supplemented with 50 ng/mL of recombinant human M-CSF (Novoprotein Scientific, Suzhou, China) and 10 ng/mL of recombinant human IL-3 (Novoprotein Scientific, Suzhou, China). The naïve iMACs were then matured in a maturation medium [RPMI1640 medium supplied with 10% (v/v) of non-heat-inactivated fetal bovine serum (FBS) (Biological Industries, Kibbutz Beit Haemek, Israel) and 50 ng/mL of M-CSF] for 2 days.