Identification of cytokines involved in hepatic differentiation of mBM-MSCs under liver-injury conditions

Figure 1 Analysis of albumin synthesis in cells for 20 d by ELISA (A_{450 nm}). MSC group: Control MSCs cultured in medium A with PBS; C group: MSCs induced with liver-injury conditioned medium with nonspecific IgG; (A) E1-E6 groups: MSCs induced with liver-injury conditioned medium and different combinations of specific antibodies (E1: Anti-FGF-3 + anti-FGF-4; E2: Anti-FGF-10 + anti-FGF-12 + anti-FGF13; E3: Anti-FGF-14 + anti-FGF-15 + anti-FGF-17; E4: Anti-FGF-18 + anti-FGF-20 + anti-FGF-21; E5: Anti-HGF + anti-OSM + anti-bNGF; E6: Anti-IGF-1 + anti-TGF-β1 + anti-TGF-β2 + anti-TGF-β3); (B) E1-E5 groups: MSCs induced with liver-injury conditioned medium and different specific antibody lonely (E1: Anti-FGF-4; E2: Anti-FGF-3; E3: Anti-bNGF; E4: Anti-HGF; E5: Anti-OSM). ^bP < 0.01 vs C group. ELISA: Enzyme-linked immunosorbent assay; MSC: Mesenchymal stem cells; PBS: Phosphate buffered saline; FGF: Fibroblast growth factor; HGF: Hepatocyte growth factor; OSM: Oncostatin M.

Figure 2 Expression analyses of FGF-4, HGF and OSM during liver injury. A-C: The kinetic expression of FGF-4, HGF and OSM mRNAs detected by real-time PCR during liver injury; D-F: The kinetic expression of FGF-4, HGF and OSM proteins detected by ELISA during liver injury. ^aP < 0.05, ^bP < 0.01 vs 0 h.

Figure 3 Detection of kinetic secretions of HGF (A), FGF-4 (B) and OSM (C) proteins in liver-injury conditioned medium. Experiment: Hepatocyte-injury conditioned medium; Control: Normal-hepatocyte conditioned medium.

Figure 4 Effects of FGF-4, HGF and OSM in hepatic differentiation. A number of depletion experiments using different combination of antibodies were conducted, and the expression of both liver-specific markers and function was analyzed. A-D: The expression of both the early liver-specific markers (HNF3β and AFP) 10 d after induction and the late liver-specific markers 20 d after induction (ALB and TAT) at mRNA levels detected by real-time PCR; E, F: The expression of AFP 10 d after induction and ALB 20 d after induction at protein levels detected by ELISA; G: Analysis of intracellular glycogen accumulation by PAS staining on day 20 after induction; H: Urea production by differentiated cells on day 20 after induction. MSC group: Control MSCs cultured in medium A with PBS; C group: MSCs induced with liver-injury conditioned medium with non-specific IgG; E1-E3 groups: MSCs induced with liver-injury conditioned medium and different concentrations of specific antibodies (E1: 1:500; E2: 1:200; E3:1:100). ^aP < 0.05, ^bP < 0.01 vs C group.

Figure 5 ALB and urea production detection on day 20 after induction in a rescue experiment in which FGF-4, HGF and OSM were added back to the cytokine-removed medium treated with anti-FGF-4 (1:100), anti-HGF (1:100) and anti-OSM (1:100). A: Analysis of albumin secretion by ELISA; B: Urea production was measured using a colorimetric assay kit. MSC group: Control MSCs cultured in medium A with PBS; C group: MSCs induced with liver-injury conditioned medium only; E1-E4: MSCs induced with liver-injury conditioned medium including anti-FGF-4 (1:100), anti-HGF (1:100) and anti-OSM (1:100), as well as different concentrations of the cytokines (E1: 0 ng/mL FGF-4 + 0 ng/mL HGF + 0 ng/mL OSM; E2: 1.25 ng/mL FGF-4 + 2.5 ng/mL HGF + 1.25 ng/mL OSM; E3: 2.5 ng/mL FGF-4 + 5 ng/mL HGF + 2.5 ng/mL OSM; E4: 5 ng/mL FGF-4 + 10 ng/mL HGF + 5 ng/mL OSM). ^aP < 0.05, ^bP < 0.01 vs C group.

Figure 6 Hepatic differentiation induced directly by different combinations of FGF-4, HGF and OSM. A: Analysis of AFP synthesis by ELISA on day 10; B: Analysis of albumin synthesis by ELISA on day 20. C group: MSCs induced with medium A and PBS; E1-E7: MSCs induced with different combinations of FGF-4, HGF and OSM (E1: OSM 10 ng/mL; E2: HGF 20 ng/mL; E3: FGF-4 10 ng/mL; E4: HGF 20 ng/mL + FGF-4 10 ng/mL; E5: HGF 20 ng/mL + FGF-4 10 ng/mL + OSM 10 ng/mL; E6: FGF-4 10 ng/mL + OSM 10 ng/mL; E7: HGF 20 ng/mL + OSM 10 ng/mL).