Culturing and Differentiating Patient-Derived FAPs

Freshly sorted FAPs were cultured in 24-well plates at an initial density of 5000 cells per well in F10 + 20% fetal bovine serum + 1% antibiotics (standard FAP media) and were grown to a density of 0.5 × 10⁶ cells per well before being passaged. Cells were passaged twice before treatment with terminal differentiation medium, which included fibrogenic media (+ 10 ng/mL TGF-β1), adipogenic media (StemPro adipogenic differentiation kit; ThermoFisher), or beige differentiation media (StemPro adipogenic differentiation kit + 10 μM amibegron) for 2 weeks. No substantial proliferation was observed after cells were switched to differentiation medium conditions. Three technical replicates were performed for each patient-derived sample. Cells were then fixed in 4% paraformaldehyde and stained for α-SMA (fibrogenic marker), perilipin (adipogenic marker), or UCP-1 (beige adipogenic marker). Expression indices for each treatment condition were quantified using ImageJ (National Institutes of Health) as previously described.^{3 (link)}