Figure 1.
Schematic presentation of the differentiation protocol.
Table 1.
Primers and Annealing Temperatures Used for RT-PCR.
Figure 2.
Characterization of human BMMSCs.
(A–C) Cells cultured in osteoblast-inductive medium for three weeks, visualized with ALP (A), alizarin red (B) and von Kossa (C) staining to show human BMMSCs that differentiated into osteogenic cells. Scale bar, 100 μm; (D) Chondrogenic differentiation in chondrocyte-inductive medium after three weeks, with the differentiated cells examined via alcian blue Staining. Scale bar, 50 μm; (E, F) Adipogenic differentiation of human BMMSCs induced by adipocyte-inductive medium. One week after induction, intracellular fat droplets were detected via phase-contrast light microscopy (E) and Oil Red O staining (F). Scale bar, 10 μm.
Figure 3.
Light microscopy of the hepatic differentiation of human BMMSCs.
(A and D) The morphological changes of differentiated cells in Groups A (VPA+) and B (VPA−) were observed under a Zeiss phase contrast microscope at days 3; (B and E) Morphological changes of differentiated cells at days 14; (C and F) Morphological changes of differentiated cells at days 21. Scale bar, 20 μm.
Figure 4.
Hepatic gene expressions of differentiated cells at the mRNA level.
(A) PCR results showing the time-dependent expression of AFP, HNF3β, and ALB in both groups during hepatic differentiation; (B) Expression level of ALB as confirmed by RT-PCR (**P<0.01 versus VPA untreated group); (C) Expression level of AFP detected by RT-PCR (**P<0.01 versus VPA untreated group).
Figure 5.
Immunofluorescence staining of hepatocyte-specific markers in the differentiated cells.
(A, B) Differentiated cells were stained with a specific antibody for HNF3β in untreated (A) and VPA-treated groups (B); (C, D) Immunofluorescence of ALB showed similar results in the control (C) and VPA-treated cells (D). Scale bar, 20 μm.
Figure 6.
Functional evaluation of differentiated cells.
(A, B) PAS staining of untreated (A) and VPA-treated (B) cells; (C, D) Expression of functionally active CYP2B in untreated (C) and VPA-treated (D) cells; (E-G) Urea production, ALB and AFP syntheses at various time points during differentiation (**P<0.01 versus VPA untreated group). Scale bar, 50 μm.
Figure 7.
Histone acetylation by immunofluorescence staining and Western blot analysis.
(A) Immunofluorescence of acetylated histones H3 and H4 in cell nuclei exposed to 5 mM of VPA for 72 h with counterstaining by DAPI; (B) Western blot analysis showing the significantly up-regulated levels of histone H3 and H4 acetylation after VPA treatment. Densitometric quantifications related to β-actin were analyzed (**P<0.01 versus 0 mM VPA group, n = 5). Scale bar, 20 μm.