ABSTRACT
Adult human dental pulp stem cells (hDPSCs) are a unique population of precursor cells those are isolated from postnatal dental pulp and have the ability to differentiate into a variety of cell types utilized for the formation of a reparative dentin-like complex. Using LC-MS/MS proteomics approaches, we identified the proteins secreted from the differentiating hDPSCs in mineralization media. Lysyl oxidase-like 2 (LOXL2) was identified as a protein that was down-regulated in the hDPSCs that differentiate into odontoblast-like cells. The role of LOXL2 has not been studied in dental pulp stem cells. LOXL2 mRNA levels were reduced in differentiating hDPSCs, whereas the levels of other LOX family members including LOX, LOXL1, LOXL3, and LOXL4, are increased. The protein expression and secretion levels of LOXL2 were also decreased during odontogenic differentiation. Recombinant LOXL2 protein treatment to hDPSCs resulted in a dose-dependent decrease in the early differentiation and the mineralization accompanying with the lower levels of odontogenic markers such as DSPP, DMP-1 and ALP. These results suggest that LOXL2 has a negative effect on the differentiation of hDPSCs and blocking LOXL2 can promote the hDPSC differentiation to odontoblasts.
Subject(s)
Adult Stem Cells/physiology , Amino Acid Oxidoreductases/metabolism , Cell Differentiation/genetics , Dental Pulp/physiology , Odontogenesis/genetics , Adult Stem Cells/drug effects , Amino Acid Oxidoreductases/genetics , Amino Acid Oxidoreductases/pharmacology , Biomarkers/metabolism , Calcification, Physiologic/drug effects , Cell Differentiation/drug effects , Cells, Cultured , Chemokine CCL27/metabolism , Dental Pulp/drug effects , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/genetics , Humans , Odontogenesis/drug effects , Phosphoproteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Sialoglycoproteins/metabolism , Stem Cell NicheABSTRACT
The differentiation of odontoblasts is initiated by the organization of differentiating ameloblasts during tooth formation. However, the exact roles of ameloblast-derived factors in odontoblast differentiation have not yet been characterized. We investigated the effects of preameloblast-conditioned medium (PA-CM) on the odontogenic differentiation of human dental pulp stem cells (hDPSCs) in vitro and in vivo. Furthermore, we analyzed the PA-CM by liquid chromatography-mass spectrometry to identify novel factors that facilitate odontoblast differentiation. In the co-culture of MDPC-23 cells or hDPSCs with mouse apical bud cells (ABCs), ABCs promoted differentiation of odontoblastic MDPC-23 cells and facilitated odontoblast differentiation of hDPSCs. PA-CM, CM from ABCs after 3 days culture, was most effective in increasing the dentin sialophosphoprotein promoter activity of odontoblastic MDPC-23 cells. When PA-CM-treated hDPSCs were transplanted into immunocompromised mice, they generated pulp-like structures lined with human odontoblast-like cells showing typical odontoblast processes. However, during recombinant human bone morphogenenetic protein 2-treated hDPSCs transplantation, some of the cells were entrapped in mineralized matrix possessing osteocyte characteristics. After proteomic analyses, we identified 113 types of proteins in PA-CM, of which we characterized 23. The results show that preameloblast-derived factors induce the odontogenic differentiation of hDPSCs and promote dentin formation.
Subject(s)
Ameloblasts/cytology , Cell Differentiation/drug effects , Culture Media, Conditioned/pharmacology , Dental Pulp/cytology , Odontogenesis/drug effects , Stem Cells/cytology , Adolescent , Ameloblasts/drug effects , Ameloblasts/metabolism , Animals , Calcification, Physiologic/drug effects , Calcification, Physiologic/genetics , Cell Differentiation/genetics , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Integrin-Binding Sialoprotein/genetics , Integrin-Binding Sialoprotein/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Odontoblasts/cytology , Odontoblasts/drug effects , Odontoblasts/ultrastructure , Odontogenesis/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Promoter Regions, Genetic/genetics , Proteomics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sialoglycoproteins/genetics , Sialoglycoproteins/metabolism , Stem Cells/drug effects , Stem Cells/metabolism , Young AdultABSTRACT
Human adult dental pulp stem cells (hDPSCs) are a unique precursor population isolated from postnatal dental pulp and have the ability to regenerate a reparative dentin-like complex. In this study, we investigated the role of Asporin in hDPSCs, which was identified as a matrix protein in our previous dentin proteomic analysis. We isolated a clonogenic, highly proliferative population of cells from adult human dental pulp. These isolated hDPSCs were confirmed by fluorescence activated cell sorting (FACS) using stem cell-specific markers and have shown multilineage differentiation potential. The localization of Asporin was identified by immunohistochemistry in the globular calcification region in the junction of predentin and dentin. The gene and protein expression levels of Asporin were enhanced at the early stage of and then reduced during the late stage of differentiation of hDPSCs in mineralization media. ASPN knock-down using a lentiviral system suppressed the mineralization of hDPSCs. These results suggest that ASPN plays positive roles in the mineralization of hDPSCs and predentin to dentin.
