Hepatocyte growth factor hgf

To generate hepatocytes from both control and patient iPSC clones, we employed an already described method that allow the efficient production of HLCs. (Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells [26]. Briefly, iPSC clones were plated on Matrigel and cultivated for 20–22 days changing the media composition as follows: 5 days in RPMI media supplemented with B27 and 100 ng/ml of Activin A (R&D), 5 days in RPMI media supplemented with 20 ng/ml BMP4 (R&D) and 10 ng/ml bFGF (Preprotech), 5 days in RPMI-B27 supplemented with 20 ng/ml hepatocyte growth factor (HGF, Invitrogen) and finally further 5–7 days in Hepatocyte Culture Medium (Lonza) supplemented with 20 ng/ml Oncostatin M (Invitrogen).

Cell banks: UC−MSCs in MCB and WCB were recovered and seeded in T75 flasks at a cell density of 2 × 10⁴ cells/cm². Twenty-four hours before cell passage, the culture medium was replaced with 10 mL fresh UC−MSC medium. When passaging cells, the culture medium was collected and centrifuged at 300× g for 5 min to remove dead cells for growth factor detection. The number of viable cells was monitored using AOPI in the automatic fluorescent cell counter to calculate the amount of factor secretion per 1 × 10⁶ cells in 24 h.
Cell sheet-forming medium: Cell sheet-forming medium was collected and centrifuged at 300× g for 5 min to remove dead cells for growth factor detection.
Cell sheet reattachment medium: The freshly produced and 24 h preserved cell sheets were attached to 100 mm Petri dishes in UC−MSC medium and cultured for 24 h. The medium was collected and centrifuged at 300× g for 5 min to remove dead cells for growth factor detection.
Hepatocyte growth factor (HGF) (Invitrogen (Carlsbad, CA, USA)), vascular endothelial growth factor (VEGF) (NOVUS (Centennial, CO, USA)), interleukin-8 (IL-8) (R&D (Minneapolis, MN, USA)), and interleukin-6 (IL-6) (NOVUS) were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturer’s instructions.