Thp 1

The 293-EBNA, 293FT, U937, THP-1, HTC-116, HT-29 cell lines were obtained from ATCC (Manassas, VA, USA). NHDF (normal human dermal fibroblasts) cells were obtained from LONZA (Basel, Switzerland). U937, THP-1, HTC-116, HT-29 cells were cultured in RPMI medium (LONZA, Basel, Switzerland) with 10% fetal bovine serum; 293-EBNA in Dulbecco’s modified Eagle medium (LONZA, Basel, Switzerland) with 10% fetal bovine serum containing 250 μg/ml G418; NHDF and 293FT cells were cultured in Dulbecco’s modified Eagle medium (LONZA, Basel, Switzerland) with 10% fetal bovine serum. All the cells were maintained at 37 ̊C under a humidified atmosphere containing 5% CO₂ and verified to be free of mycoplasma contamination using the MycoAlert™ Mycoplasma Detection kit (LT07–318, LONZA).

The monocyte phenotype after co-incubation with hMSC CM was analyzed using the human monocytic leukemia cell line (Thp-1; Sigma, Switzerland). Thp-1 cells were cultured in suspension using xVivo15 medium (Lonza, Switzerland) and the medium was changed every 2–3 days. Thp-1 cells were co-incubated for 16 h with either (I) hMSC CM (n = 5 per hMSC group), (II) xVivo15 (n = 5) or (III) basal DMEM (n = 5). 0.2 ml hMSC CM diluted 1:3 in basal DMEM was used per 100’000 Thp-1. After co-incubation, floating and attached Thp-1 were harvested using Accutase and stained 20 min at 4 °C with CD11b, CD14, CD16, CD36, and SRA-I (supplementary Table S2). All events were acquired using a LSR Fortessa and the data sets were analyzed by FlowJo software gating viable and single cells (for gating strategy see supplementary Fig. S13). Bidimensional t-SNE maps were generated from multidimensional flow cytometry data (all events included) and PhenoGraph algorithm employed.