Transforming growth factor beta tgfβ

The isolated monocytes were cultured for 5 days in RPMI 1640, supplemented with 10% FBS and 25 ng/ml of Recombinant Macrophage Colony-Stimulating Factor (M-CSF, PeproTech, Rocky Hill, USA) to generate M0 macrophages. After 5 days, macrophages were polarized in vitro toward M1 or M2 phenotypes. For M1-like polarization, macrophages were cultured in RPMI-1640 medium supplemented with 100 ng/ml lipopolysaccharides (LPS from E. coli; Sigma-Aldrich) and 50 ng/ml interferon-gamma (IFN-γ; PeproTech) and incubated for 18 h. Meanwhile, for M2-like polarization macrophages were cultured in a complete RPMI-1640 medium supplemented with 20 ng/ml interleukin-4 (IL-4; PeproTech), for 18 h.
The culture medium of M2 macrophages was supplemented with tumor necrosis factor-alpha (TNFα; 20 ng/ml; PeproTech) and Transforming Growth Factor-beta (TGFβ; 10 ng/ml; PeproTech) for 18 h as previously reported [18 (link)].

Differentiation of murine Th1 and Th17 cells was performed as previously described [34 (link), 35 (link)]. Briefly, for Th1 cell differentiation, naïve CD4⁺ T cells were purified from spleens by using a naïve CD4⁺ T cell Isolation Kit (Stem Cell Technologies, Vancouver, BC, Canada) and were cultured in 2.5 μg/mL anti-CD3 and 5 μg/mL anti-CD28 (eBioscience) pre-coated culture plates with 20 ng/mL IL-2 (PeproTech), 20 ng/mL IL-12 (PeproTech), and 5 μg/mL anti-IL-4 (eBioscience) for stimulation. For Th17 cell differentiation, naïve CD4⁺ T cells received the same treatment as for Th1 differentiation except 20 ng/mL IL-6 (PeproTech), 3 ng/mL transforming growth factor-beta (TGF-β) (PeproTech), 20 ng/mL IL-23 (PeproTech), 5 μg/mL anti-IFN-γ (eBioscience), and 5 μg/mL anti-IL-4 (eBioscience) were used for stimulation instead. To confirm the role of Gal-9 in UC-MSCs, the cells were treated with UC-MSCs alone or UC-MSCs plus 10.8 mg/mL α-lactose (Sigma-Aldrich). After culture for 72 h, the cells were collected for further analysis.