Mesenchymal stem cell basal medium

Explanted lung tissue samples were obtained from patients with genetically confirmed HPS‐1 who underwent clinically indicated lung transplantation for HPSPF. Lung fibroblasts were cultured in growth media containing 10% Mesenchymal Cell Growth Supplement, 2% L‐Glutamine, and 0.1% Gentamicin Sulfate in Lonza's Mesenchymal Stem Cell Basal Medium (Lonza, Switzerland). Control lung fibroblasts were purchased from Lonza and maintained in the same growth media. When the fibroblasts reached 80–90% confluency, cells were incubated with either 1 μM MRI‐1867, 1 μM Rimonabant, 1 μM 1400W, or DMSO for vehicle and fibrosis‐only control. One hour later, the cells were stimulated with 10 ng/ml TGFβ‐1 (240‐B‐010, R&D Systems) diluted in 4 mM HCL in 1 mg/ml BSA (R&D Systems), while the vehicle and fibrosis‐only control were incubated with equal volume of diluent. Twenty‐four hours later, culture supernatant was collected and rapidly frozen for cytokine and endocannabinoid measurement. Cells were washed three times with PBS, trypsinized, washed, pelleted, and frozen. RNA was collected using Maxwell® RSC simplyRNA Cells Kit (AS1390) on a Maxwell® RSC Instrument (AS4500, Promega). RNA quality and quantity were measured using QuantiFluor® RNA System (E3310) on a Quantus™ Fluorometer (E6150, Promega). One microgram of RNA was transcribed to cDNA as above.