ABSTRACT
Objective To explore the effect and mechanism of Chir99021 on osteogenic differentiation of rat dental pulp stem cells. Methods Primary rat dental pulp stem cells were isolated from rat dental pulp and verified by fluorescence immunoassay. Different concentrations of Chir99021 were set, and the cell proliferation was detected by CCK⁃8 to select the optimal concentration. Osteogenic differentiation was detected by alizarin red staining. The expression of osteogenic differentiation related genes and proteins recombinant wingless type MMTV integration site famity member 1 (Wnt1), Wnt3a and Wnt3a β⁃expression of catenin, axis inhibition protein 2(Axin 2), dentin sialophosphoprotein(OCN) and dentin matrix acidic phosphoprotein 1(DMP1) was detected by Real⁃time PCR and Western blotting. Results The positive expression of dentin sialophosphoprotein (DSPP) and vimentin indicated that rat dental pulp stem cells were successfully isolated. After osteogenic induction of rat dental pulp stem cells, calcium deposits significantly increased with the addition of glycogen synthase kinase⁃3β(GSK⁃3β) inhibitor Chir99021, calcium deposits were significanted reduced. After osteogenic differentiation of rat dental pulp stem cells, the expression of Wnt1, Wnt3a, β⁃catenin, Axin2, OCN and DMP1 increased, while the expression of Wnt1, Axin2, OCN and DMP1 decreased with the addition of Chir99021. Conclusion Chir99021 can inhibit the osteogenic differentiation of rat dental pulp stem cells after 7 days of induction.
ABSTRACT
Objective To investigate the effects of CHIR99021 and Wnt3a, Wnt/β-catenin signaling pathway acti-vators, on cardiac differentiation of mouse embryonic stem cells ( mESCs ) . Methods The embryonic bodies ( EBs) were formed through suspension culture method, CHIR99021 or Wnt3a was added into differentiated medi-um from day 2 to 5, named CHIR99021 group or Wnt3a group, respectively. In addition, there was a control group in which EBs were automatically differentiated. The expression levels of Brachyury, the mesoderm specific target gene, and Nkx2. 5, cardiac-precursor marker, as well as the transcripts of cardiomyocyte markers,α-myosin heavychain (α-MHC ) , cardiac troponin T ( cTnT ) and connexin-43 ( Cx43 ) were analyzed through quantitative RT-PCR. Besides, the cardiac-specific proteins including α-MHC, cTNT and CX43 were detected by immunofluores-cence and Western blot. Results The mESCs in every group did differentiate into cardiomyocytes. The expression of Brachyury was substantially augmented by treatment with CHIR99021 and Wnt3a, showing a peak of expression at day 7. Similarly, CHIR99021 and Wnt3a dramatically increased the expression levels of Nkx2. 5,α-MHC, cT-nT and Cx43 with the time of differentiation, with the expression of target genes in CHIR99021 group and Wnt3a group was greater than that in the control group and CHIR99021 group was higher than Wnt3 a group at day 15 ( P<0. 05, P < 0. 01 ). Western blot analysis suggested that the expressions of α-MHC, cTNT and CX43 in CHIR99021 group and Wnt3a group were greater than those in the control group, and CHIR99021 group was higher than Wnt3 a group at day 15 . Conclusion Both CHIR99021 and Wnt3 a could improve cardiogenesis from mESCs through activate Wnt/β-catenin signaling pathway at the early stage of differentiation while the former is better than the latter.