Fig. 2

NBIF-fed hBMSCs have greater osteogenic differentiation capacity with distinct molecular changes. a, b Representative osteogenic induction assay images (a) and quantification results (b) of hBMSCs. The BMSC were first cultured in FBS- (column 1) or NBIF- (columns 2 and 3) supplemented medium for 7 days and then subjected to osteogenic induction (OI) for 7, 14 and 21 days in the presence of FBS (columns 1 and 2) or FBS plus NBIF (column 3), followed by Alizarin Red staining. Data in (b) [as well as in (c, d, f–h)] were expressed as mean ± standard error of the mean (SEM) of the fold change across three replicates for each group. P-values were obtained from an unpaired t-test; **P ≤ 0.01, ***P ≤ 0.001. Scale bar = 100 μm. c, d RT-PCR detection of expression levels of the osteogenesis-related genes RUNX2 (c) and COL1A1 (d) after 28-day osteogenic induction. e, f Representative images and quantification data of adipogenic differentiation of hBMSCs. The hBMSCs were first cultured in FBS or NBIF-supplemented medium for 7 days and then in adipogenic differentiation medium for 28 days, followed by Oil red staining. Scale bar = 100 μm. g, h RT-PCR detection of expression levels of the adipogenesis-related genes PPARG (g) and FABP4 (h) after 28-day of adipogenic induction. i Represent images of Alcian Blue staining to indicate Chondrogenesis. j Unsupervised principal component analysis (PCA) of RNA-seq data based on gene transcription profiles from the indicated hBMSC samples. MSCs cultured in NBIF and those cultured in FBS distinctly clustered within the second principal component (PC2), which explained 42% of the observed variation. k, l Gene Ontology (GO) analysis for upregulated (k) and downregulated (l) genes in hBMSCs cultured in NBIF-supplied medium compared to FBS-supplied medium. The top 20 enriched biological processes were presented. Scale bar = 500 μm