Oncostatin m osm

ADHLSCs at passage 5 or 6 were seeded at a density of 1 × 10⁴ cells/cm² onto collagen I-coated 175 cm² flasks in complete DMEM medium. Twenty-four hours later, the culture medium was switched to IMDM (Thermo Fisher Scientific). Cells were then subjected to a four-step differentiation protocol as previously described [39 (link)]. First, cells were incubated for 2 days with IMDM containing 20 ng/ml epidermal growth factor (EGF) (PeproTech) and 10 ng/ml basic fibroblast growth factor (bFGF) (PeproTech). Then, the cells were incubated for 10 days with IMDM containing 20 ng/ml hepatocyte growth factor (HGF) (PeproTech), 10 ng/ml bFGF, nicotinamide 0.61 g/l (Sigma Aldrich), and 1% insulin-transferrin-selenium (ITS) (Invitrogen) premix. Next, the cells were incubated for 10 days with IMDM containing 20 ng/ml HGF, 20 ng/ml oncostatin M (OSM) (PeproTech), 0.61 g/l nicotinamide, and 1% ITS premix. Finally, the cells were treated with IMDM containing 20 ng/ml OSM, 1 μM dexamethasone (Sigma Aldrich), and 1% ITS premix for 10 days. For each step, the medium was changed every three days. Negative controls were performed using IMDM supplemented with 1% FCS and 1% of Penicillin/Streptomycin. Cells were harvested either after each step or at the end of the differentiation protocol and used for MLR, RT-PCR, fluorescence microscopy, or flow cytometry analysis.

Stem cells were differentiated by the previously published conventional hepatogenic differentiation protocol [9 ] and an advanced protocol. Briefly, in the advanced protocol, the cells were seeded on 0.1% gelatin-coated dishes at 7000 cells/cm² in a cell culture medium. After 2 days, they were cultured for 7 days with Step-1 medium consisting of Iscove’s Modified Dulbecco’s Medium (IMDM; Gibco) supplemented with 0.1% polyvinyl alcohol (PVA; Sigma Aldrich) or 1% FBS, 10 mM nicotinamide (Sigma Aldrich), 20 ng/ml hHGF (Peprotech), 10 ng/ml FGF2, 2 μM 5-azacytidine (Sigma Aldrich), 0.1 μM dexamethasone (Sigma Aldrich), 1% insulin-transferrin-selenium (ITS; Gibco), 3 μM CHIR99021, 20 ng/ml EGF (Peprotech), and 10 μM Fasudil (AdooQ Bioscience, Irvine, CA, USA). For hepatic maturation, the Step-1 medium was replaced with Step-2 medium consisting of IMDM supplemented with 1 μM dexamethasone, 1% ITS, 20 ng/ml Oncostatin M (OSM, Peprotech), 20 ng/ml hHGF, and 10 uM Fasudil.

When the cells attained a confluence of 40%, mTeSR™1 was replaced with RPMI 1640 (Gibco), containing 2% B27 minus insulin (Gibco), 100 ng/mL activin A (Peprotech), and 50 ng/mL Wnt 3a (R&D System) for 3 days. The medium was then replaced with IMDM (Procell) with 20% knockout serum replacement (Gibco), 1% GlutaMAX™ supplement (Gibco), 1% dimethyl sulfoxide (DMSO) (Sigma-Aldrich), 1% nonessential amino acids (NEAA) (Gibco), and 0.1 mM β-mercaptoethanol (β-ME) (Sigma-Aldrich) for 4 days. Next, the cultures were placed in IMDM containing 1% GlutaMAX™ supplement, 20 ng/mL oncostatin M (OSM) (Peprotech), 5 ng/mL basic fibroblast growth factor (bFGF) (Peprotech), 0.5 μM dexamethasone (Dex) (Sigma-Aldrich), and 1% insulin-transferrin-selenium (ITS) (Sigma-Aldrich) for 5 days. Finally, the differentiated cells were cultured in hepatocyte culture medium (HCM) (Lonza) supplemented with 10 ng/mL hepatocyte growth factor (HGF) (Peprotech), 0.5 μM Dex, 10 μM lithocholine acid (LCA) (Sigma-Aldrich), 10 μM vitamin K₂ (Sigma-Aldrich), and 1% ITS for continuous cultivation.

hASCs were plated at a density of 40,000 cells/cm² on collagen I (Invitrogen)-coated dishes (Nunc, Roskilde, Denmark) and cultured in DMEM/F-12 (Invitrogen) supplemented with 10% FBS-MSCS (HyClone), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO₂. Once the cells reached 90% confluence, they were washed twice with PBS and incubated with serum-free DMEM/F-12 medium for 48 hours.
To induce definitive endoderm differentiation, the cells were incubated with DMEM/F-12 containing 0.5 mg/mL albumin fraction V(BSA) (Sigma-Aldrich), 50 ng/mL Wnt3a (Peprotech, Rocky Hill, NJ,USA), or GSK3 inhibitors Chir98014 (0.2 μM, Selleckchem, Houston, Texas, USA), or Chir99021 (2 μM, Selleckchem) for 24 hours, or 100 ng/mL activin A (Peprotech) for 72 hours; 1% ITS (Sigma-Aldrich) was added to the medium beginning at the second day.
For subsequent hepatic differentiation, the medium was changed to MEM/NEAA (Invitrogen), supplemented with 0.5 mg/mL BSA, 1% ITS, 20 ng/mL BMP2 (Peprotech) and 30 ng/mL FGF4 (Peprotech) for 5 days. To allow for hepatocytes maturation, the cells were further treated with 20 ng/mL Hepatocyte Growth Factor (HGF) for 5 days, and 20 ng/mL HGF, 10 ng/mL oncostatin M (OSM) (Peprotech) plus 10⁻⁶ M Dexamethasone (DEX, Sigma-Aldrich) treatment for another 5 days. The differentiation media were changed every 2 days.

hADSCs between passage 3 and 7 were planted at a density of 2–3 × 10⁴ cells/cm² on collagen I-coated dishes (Invitrogen) and cultured in expansion medium at 37 °C with 5 % CO₂. Once the cells reached 100% confluence, they were incubated with 10 % FBS RPMI-1640 (Gibco) medium containing 1 μM ATRA for 24 h. The cells were then incubated with serum-free RPMI-1640 medium containing 100 nM IDE1, 3 μM CHIR99021, and 10 μM LY294002 (Selleckchem) for 24 h. Next, the cells were then incubated with serum-free RPMI-1640 medium containing 100 nM IDE1, 10 μM LY294002, 250 nM LDN-193189, and 20 ng/mL FGF4 (PeproTech) for 2 days and then changed to serum-free RPMI-1640 medium containing 100 nM IDE1, 10 μM LY294002, and 20 ng/mL FGF4 for 24 h. The medium was changed to Williams’ E (Gibco) supplemented with 150 ng/mL hepatocyte growth factor (HGF; Sino Biological), 20 ng/mL FGF4, 30 ng/mL oncostatin M (OsM; PeproTech), 2 × 10⁻⁵ mol/L dexamethasone (Dex, Sigma-Aldrich), and 1% ITS (Sigma-Aldrich). The differentiation medium was changed every 2 days.

Human primary KCs were collected from the foreskins of twelve patients (aged 8 to 30 years) who underwent urological surgery at the Department of Urology, Xijing Hospital, as previously described 33 (link). Keratinocyte complete media (EpiLife, Thermo Fisher Scientific, USA) supplemented with EpiLife medium +60 µM calcium/EpiLife Defined Growth Supplement (EDGS, Thermo Fisher Scientific, USA) was used to sustain human primary KCs. Penicillin‒streptomycin (50 U/mL) was added. Human primary KCs were then treated with ENO1 siRNA, ENOBlock, BI-D1870, K17 and mutant K17 overexpression plasmids in 6-well plates.
The human HaCaT keratinocyte cell line was purchased from KeyGEN Biotech (GB300, Nanjing, China), cultured in Dulbecco's Modified Eagle's medium (DMEM; Gibco-Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-Invitrogen, Carlsbad, USA) and maintained at 37°C in a humidified environment with 5% CO₂. After 24 h of starvation, cells at 40-60% confluence were stimulated with IL-17, IL-22, tumor necrosis factor-α (TNF-α), IL-1α and oncostatin M (OSM) (50, 20, 50, 20 and 20 ng/mL, respectively) (PeproTech, Rocky Hill, NJ, USA) for 48 h, and PBS treatment served as a negative control.