Fig 1.
Establishment of a tetracycline (Tet)-controlled Amelx expression system in MSCs.
(a): Schematic diagram depicting the procedure to establish a Tet-controlled Amelx expression system in MSCs. TetR: Tet repressor, TO: Tet operator, Dox: doxycycline (tetracycline derivative). (b): MSC colony (MSC-TetR) in culture medium containing 500 μg/mL geneticin 10 days after transduction with the pLenti3.3/TetR expression vector (bar; 60 μm). (c): Expression of TetR repressor gene in MSCs (without transduction) and MSCs-TetR was determined by RT-PCR. HEK293 cells subjected to the same transduction procedure (HEK-TetR) were used as a positive control. (d, e): MSCs-TetR were lentivirally transduced with the expression vector pLenti6.3/TO/V5/Amelx or plenti6.3/V5-GW/GFP. MSCs-TetR/Amelx (d) and MSCs-TetR/GFP (e) were selected by 10 μg/mL blastcidin S (bar; 200 μm).
Table 1.
Primers used for RT-PCR analyses.
Table 2.
Primers used for quantitative real-time RT-PCR analyses.
Fig 2.
Induction of Amelx expression in MSCs-TetR/Amelx by Dox treatment.
MSCs-TetR/Amelx were cultured in the growth medium in the presence (+) or absence (-) of Dox for 24–72 hours. Inducible expression of the Amelx gene was detected by RT-PCR (a) and western blot (b) analyses. GAPDH was used as a loading control. (c) MSCs-TetR/Amelx were cultured in the osteogenic induction medium in the presence (+) (black bars) or absence (-) (white bars) of Dox for 17 days in four different conditions. (Condition I: day 0–17 Dox-; Condition II: day 0–14 Dox-, day 15–17 Dox+; Condition III: day 0–14 Dox+, day 15–17 Dox-; Condition IV: day 0–17 Dox+). Expression of Amelx was determined by quantitative real-time RT-PCR analysis. Significant differences (*P<0.01: ANOVA with Tukey’s multiple comparison test: n = 4) within each condition are shown.
Fig 3.
Controllable expression of osterix, BSP and osteocalcin in MSCs-TetR/Amelx by Dox treatment.
MSCs-TetR/Amelx were cultured in the osteogenic induction medium in the presence (+) (black bars) or absence (-) (white bars) of Dox for 17 days in four different conditions. (Condition I: day 0–17 Dox-; Condition II: day 0–14 Dox-, day 15–17 Dox+; Condition III: day 0–14 Dox+, day 15–17 Dox-; Condition IV: day 0–17 Dox+). Expression of Runx2 (a), osterix (b), BSP (c), osteopontin (d) and osteocalcin (e) was determined by a quantitative real-time RT-PCR analysis. Significant differences (**P<0.01, *P<0.05: ANOVA with Tukey’s multiple comparison test: n = 3) within each condition are shown.
Fig 4.
Effects of forced expression of Amelx in MSCs-tetR/Amelx on expression of osteogenic marker genes.
MSCs-TetR/Amelx were cultured in growth medium (Con) or osteogenic induction medium (Os) in the presence (+) or absence (-) of Dox for 21 days. (a) The expression of osteogenic marker genes (Runx2, osterix, osteocalcin, BSP, and osteopontin) was examined by RT-PCR analysis. GAPDH was used as an internal control. Quantitative real-time RT-PCR analysis was performed to examine expression of osterix (b), type I collagen (c), BSP (d), and DMP1 (e) genes. GAPDH was used as an internal control. Significant differences (*P<0.01: ANOVA with Tukey’s multiple comparison test: n = 3) are shown.
Fig 5.
Effects of forced expression of Amelx on ALP activity and mineralized nodule formation.
MSCs-TetR/Amelx at 3 passages were cultured in the growth medium (Con) or osteogenic induction medium (Os) in the presence (+) or absence (-) of Dox in 24-well plates. (a) ALP activity on day 7 and 14 was examined by ALP staining (bars: 100 μm). (b) Mineralized nodule formation on day 21, 28 and 35 was detected by von Kossa staining. (c, d): Calcium deposition was determined by Alizarin Red S staining on day 7, 14 and 21 (c) and the staining intensity was quantitatively analyzed (d). Significant differences (*P<0.01: ANOVA with Tukey’s multiple comparison test: n = 9) are shown.
Fig 6.
Effects of forced expression of Amelx at different osteogenic differentiation stages on matrix calcification of MSCs.
MSCs-TetR/Amelx at 8 passages were cultured in the growth medium (Con) or osteogenic induction medium (Os). Dox was applied to MSCs-TetR/Amelx on day 0–10 (a: early stage), day 10–20 (b: intermediate stage), or day 20–30 (c: late stage) of osteogenic differentiation, and Alizarin Red S staining was performed at day 10, day 20, or day 30, respectively. The staining intensity was also quantitatively analyzed. Significant differences (*P<0.01: ANOVA with Tukey’s multiple comparison test: n = 9) are shown.