Fig. 4: Effect of omega-3 PUFAs on stem cell properties of primary BMSCs.

Analysis of stem cell phenotype by (a) colony-forming units-fibroblast (CFU-f) analysis of freshly isolated BMSCs with (b) representative pictures of CFU-f analysis (scale bar 20 mm). (n = 2–4 from pooled samples; t test *p ≤ 0.05: HFD vs HFD + F) Data are presented as mean values ± SEM. c Representative pictures of Oil red O (ORO) stained lipid droplets in AD differentiated BMSCs (scale bar 500 µm; 10x magnification). d, e Gene expression profile of BMSCs differentiated towards adipocytes in D10. d Gene expression of adipogenic genes (Ppary2, Cebpa, Adipoq, Cd36, Fsp27, Vcam) and (e) oxidative stress, senescence and inflammatory genes (Sod2, Hmox1, p53, Tnfα) (n = 2 from pooled samples). Data are presented as mean fold change (F.C.) of gene expression normalized to BMSCs from ND group ± SEM. f Representative pictures of Alizarin Red (AZR) staining for calcified matrix mineralization of OB differentiated BMSCs (scale bar 500 µm; 10x magnification) and (g) quantification of eluted AZR staining of mineralized matrix in OB differentiated BMSCs in D10. (n = 2–4; one-way ANOVA, Tukey’s multiple comparison test with *p ≤ 0.05. Data are presented as mean fold change (F.C.) of A₅₀₀ in OB differentiated cells (OB) normalized to BMSCs from ND group ± SEM. h Measurement of alkaline phosphatase (ALP) activity normalized to cell viability in OB differentiated BMSCs in D7. (n = 9–11; one-way ANOVA, Tukey’s multiple comparison test with **p ≤ 0.01, ***p ≤ 0.001). Data are presented as mean fold change of ALP activity in OB differentiated cells (OB) from each experimental group ± SEM. i Gene expression profile of osteoblastic markers (Alpl, Oc, Col1a1, Bmp2, Ctnnb1) in OB differentiated BMSCs in D10 (n = 2 from pooled samples). Data are presented as mean fold change (F.C.) of gene expression normalized to BMSCs from ND group ± SEM (groups coding: white column-ND, black line shading-HFD, yellow column-HFD + F).