Pbm 2

Human subcutaneous tissue stem cells (adipose-derived stem cells [ADSC; Lonza]) from 2 healthy adult donors were maintained in ADSC growth medium (ADSC-GM; Lonza). At low passage (passages 2–4), cells were seeded in 12-well plates (1 × 10⁵ cells/well) and incubated in growth medium (preadipocyte basal medium-2 [PBM-2]; Lonza), followed by adipocyte differentiation medium (PBM-2/PDM; Lonza), for 10 days. Cells displaying characteristic adipocyte morphology by phase-contrast microscopy were incubated in media with 5 ng/ml of human recombinant TGFβ2 (PeproTech) for up to 72 hours. Experiments were performed in triplicate and repeated at least 3 times with consistent results. In other experiments, fibroblasts were explanted from the interscapular skin of AdipoP-Cre+;tdTomato^+/f–transgenic mice, and low-passage confluent cultures were incubated with TGFβ2 (5 ng/ml), rosiglitazone (10 μM; Cayman Chemical), or bleomycin (1 μM; APP Pharmaceuticals) for 7 days.

All experiments were performed with extended lifespan Normal PreADipocytes (NPADs) developed from primary human preadipocytes that were derived from the subcutaneous fat tissue of a non-diabetic donor as recently described (Vu et al., 2013 (link)). The NPADs were cultured as a monolayer in Preadipocyte Basal Medium 2 (PBM-2) (Lonza, MD) supplemented with 10% fetal bovine serum (FBS), L-glutamine, gentamycin, and amphotericin according to the manufacturer’s instructions. This media is referred to as preadipocyte growth media 2 (PGM-2). For differentiation, the NPADs were seeded into 35 mm tissue culture plates at 30,000 cells/plate and allowed to grow in PGM-2 until confluent (usually 5–6 days). The cells were then induced to differentiate into adipocytes with differentiation medium consisting of PGM-2 plus dexamethasone, 3-isobutyl-1-methyl-xanthine, indomethacin, and extra insulin prepared according to the manufacturer’s instructions (Lonza, MD). The cells were left in the differentiation medium for 11 days until full development of lipid droplets occurred. Comparative control groups of confluent NPADs (preadipocytes) were cultured in normal PGM-2 without any differentiation factors for the duration of the normal time required for differentiation.