Differentiation Of Odontoblast-like Cells From Mouse Induced Pluripotent Stem Cells In Vitro

Objectives:To clarify the effect of dentin non-collagen protein(DNCP)on the differentiation of odontoblast-like cells from mouse induced pluripotent stem(miPS)cells and to explore the influence as well as possible mechanism of bone morphogenetic protein in the induced process.Methods:Part one: The miPS(OSKM cell lines)cells were co-cultured with mouse embryo fibroblast(MEF)feeder cells.The medium was miPS cell basic media.Then miPS cells were developed into embryoid bodies(EB)by drop-suspension culture for 3days and following suspension culture in non-adherent culture dishes.The medium was miPS cell basic media without LIF.Cell morphological characteristics were observed under a inverted microscope.At 2nd day after suspension culture,immunofluorescence assays were performed to detect the expression of the signature gene of pluripotent stem cells,Oct-4 and Sox-2.Part two: DNCP was extracted from the isolated teeth of mice.MTT assays were performed for assessment of cell viability in the presence of different concentrations of DNCP,100ng/ml,500ng/ml,1000ng/ml,10000ng/ml.Then EBs were dvided into four setted groups for adherent culture: the control group,the DNCP induced group,Noggin suplied DNCP induced group and BMPs suplied DNCP induced group.At10 th day after miPS cells treated with specific medium,the mRNA expression of the odontoblast markers dentin matrix protein-1(DMP-1),dentin salivary phosphoprotein(DSPP)and Msx-1 gene associated with BMP/Smad signaling way were detected by qRT-PCR.The expression level of Smad4/ p-Smad4 and p38/ p-p38 were examined by western-blot.Results:Co-culture with MEF,miPS cells were grown in clones and clones displayed thesmall and round shape.Cells in the clones were the same size and shape relatively.Furthermore,cells lined up tightly.EBs formation were achieved after drop-suspension cultured for 3 days and suspension cultured for 2 days.The EBs displayed typical state: scattered distribution,homogeneous form,large volume,solid,and no cell atypia was observed.The result of immunofluorescence demonstrated there were Oct-4 and Sox-2 expressions of EBs.MTT assays showed the mi PS cells treated with 500ng/mL DNCP had the best viability and proliferation ability in comparison with other concentrations(P < 0.05).QRT-PCR assays detected the positive expression of DMP-1and DSPP after miPS cells cultured in the microenvironment conditioned with DNCP.The mRNA level of DMP-1 and DSPP were decreased after treated with Noggin compared to DNCP group(P < 0.05).Furthermore,when exogenous BMPs was added to the medium,the expression of both genes were significantly increased(P < 0.05).The mRNA expression of Msx-1 in the media supplied with BMPs was highest among four groups and decreased in the Noggin group which was consistent with the trend of the change of DSPP and DMP-1.Western blot analysis showed a up-regulation of the expression of p38/p-p38 and Smad4/p-Smad4 in the DNCP group compared with the control group(p < 0.05).The expression of those signal proteins degraded after treated with Noggin.Moreover,the expression of p38 and Smad4 were significantly increased compared with DNCP group(P < 0.05),and the activation level of both was also increased(P < 0.05).Conclusions:The miPS cells could differentiate into odontoblast-like cells in the microenviroment conditioned with 500ng/ml DNCP.In addition the BMP/Smad and BMP/p38 signaling pathways played an important role in the induced process.