Hepatocyte growth factor (hgf)

Human melanoma lines (A375, mel-537, mel-624, mel-888, SKMEL-5, SKMEL-23, and SKMEL-28), HEK293T, and 3T3 cells (ATCC, Manassas, VA and University of Chicago Comprehensive Cancer Center Core Facilities) were cultured in DMEM/10% FBS (Sigma-Alrich, St. Louis, MO, USA). Morphology, growth curve analysis, and melanoma-marker testing verified melanoma cell identity. All cells were negative for the presence of mycoplasma. For Hepatocyte Growth Factor (HGF) treatment, cells were grown in serum-free media, supplemented with 0.5% bovine serum albumin (BSA) and 30 ng/ml HGF (EMD-Millipore, Temecula, CA) for 7.5–10 minutes prior to cell collection and analysis. For PD184352 (Sigma-Alrich) treatment, 1 μM was added for the length of the experiment.

MEFs were converted to iHeps as in (Sekiya and Suzuki, 2011 (link)). Briefly, MEFs were prepared from E13.5 embryos and serially transduced with Foxa1 and Hnf4α or Hnf4α-2ta-Foxa1 retroviruses over a five-day period, followed by culture on gelatin for two weeks in hepato-medium (DMEM:F-12, supplemented with 10% FBS, 1mg/ml insulin (Sigma), 10⁻²⁷M dexamethasone (Sigma-Aldrich), 10mM nicotinamide (Sigma-Aldrich), 2mM L-glutamine, 50mM β mercaptoethanol (Life Technologies) and penicillin/streptomycin, containing 20ng/ml hepatocyte growth factor (Sigma-Aldrich) and 20ng/ml epidermal growth factor (Sigma-Aldrich), after which the emerging iHeps were cultured on collagen. iHeps generated with mono- and bicistronic constructs were equivalent.

Mouse Embryonic Fibroblasts were derived from the C57BL/6J strain (RRID:IMSR_JAX:000664). All animal procedures were based on animal care guidelines approved by the Institutional Animal Care and Use Committee. Mouse embryonic fibroblasts were reprogrammed to iHeps/iEPs, as in Sekiya and Suzuki (2011) (link). Briefly, fibroblasts were prepared from E13.5 embryos and serially transduced with polyethylene glycol concentrated Hnf4α-t2a-Foxa1, followed by culture on gelatin for two weeks in hepato-medium (DMEM:F-12, supplemented with 10% FBS, 1 mg/ml insulin (Sigma-Aldrich), dexamethasone (Sigma-Aldrich), 10 mM nicotinamide (Sigma-Aldrich), 2 mM L-glutamine, 50 mM β-mercaptoethanol (Life Technologies), and penicillin/streptomycin, containing 20 ng/ml hepatocyte growth factor (Sigma-Aldrich), and 20 ng/ml epidermal growth factor (Sigma-Aldrich)), after which the emerging iEPs were cultured on collagen and passaged twice per week for three months.

We adapted the culturing method from Ogawa et al., 2015 and Okabe et al., 2009 (link). Briefly, 70% Matrigel in DMEM was added as a bottom layer to the plate, 96-well glass-bottom plate (20 μl) or glass-bottom 35mm μ-Dish (100 μl; iBidi) or 6-well plate (100 μl). The bottom layer was allowed to solidify at 37°C for 30 minutes. Long-term iEPs were dissociated using 0.05% Trypsin-EDTA (diluted from 0.25%; Gibco, Cat #: 25200056). The cells were resuspended in pre-chilled OVM-medium (William’s E medium, supplemented with 10% FBS, dexamethasone, 10 mM nicotinamide, 2 mM L-glutamine, 0.2 mM ascorbic acid, 20 mM HEPES, 1% penicillin/streptomycin, 1% sodium pyruvate, 0.15% of 7.5% sodium bicarbonate, 14 mM glucose, containing 1x ITS-X (Gibco), 20 ng/ml hepatocyte growth factor (Sigma-Aldrich), and 20 ng/ml epidermal growth factor (Sigma-Aldrich)). The top layer was prepared with 40% Matrigel, with 1.2mg/ml Collagen Type I (Gibco, stock of 3mg/ml), mixed with 20k cells for each well of 96-well plate, or 80k for each well of a 6-well plate. After 30 minutes, the top layer was added to the plate and allowed to solidify and set in the incubator for 45 minutes. After the top layer solidified, pre-warmed OVM medium was added. The medium was changed every other day. After five days of gel culture, the cells were imaged and processed for single-cell RNA-sequencing.