OBJECTIVE: Stem cells from human exfoliated teeth (SHED) were sorted by magnetically activated cell sorting (MACS) technique to obtain the CD146 positive and negative cell subpopulation. Then the biological characteristics of these subpopulations were compared to explore their specific application potential in tissue engineering. METHODS: In this study, freshly extracted deciduous teeth without any caries or dental pulp disease were obtained. SHED was isolated using enzyme digestion method and then sorted by MACS, CD146 positive cells and CD146 negative cells were obtained after cell sorting. The biological characteristics of the unsorted mixed cells, CD146 positive subpopulation and CD146 negative subpopulation were compared. The proliferation ability was detected through cell counting kit-8 (CCK-8) and colony-forming unit (CFU). After osteogenic induction, alizarin red staining was performed and the gene expression of osteogenic related markers was detected by quantitative real-time polymerase chain reaction(qPCR). After adipogenic induction, oil-red O staining was performed and the gene expression of adipogenic related markers was detected. After neurogenic differentiation induction, the expression of neural markers was detected by immunofluorescence and the gene expression of neural markers was detected by qPCR. RESULTS: SHED of the fifth passage was sorted by MACS. And the CD146 positive cell subpopulation and CD146 negative cell subpopulation were obtained. CCK8 assay showed that the proliferative tendency of the three cell groups was consistent, but the proliferation potential of CD146 positive and negative cell subpopulations was significantly lower than that of the unsorted cells. The colony forming rates of the unsorted mixed cell group, CD146 positive and negative populations were 28.6%±3%,17.1%±2.3% and 27.5%±2.5%, respectively. After 21 days of osteogenic induction, alizarin red staining and qPCR showed that the CD146 positive cell population had more mineralized nodule formation and expressed higher level of osteogenic related genes compared with the other two groups. After 21 days of adipogenic induction, oil red O staining and qPCR results showed that the CD146 negative subpopulation produced more lipid droplets and the expression of lipid related genes increased more significantly. After 14 days of neural induction, cell immunofluorescence and qPCR results showed that the unsorted mixed cell group and CD146 positive subpopulation expressed glial cell marker, and the expressions of neural precursor cells and neuronal marker increased significantly in negative subpopulation. CONCLUSION The unsorted mixed cells showed better proliferative potential than CD146 positive and negative subpopulations. The CD146 positive subpopulation was most potent in osteogenic differentiation; it was more suitable for bone tissue engineering. The CD146 negative cells had stronger adipogenic differentiation potential than the other two cell groups; different subpopulations differed in neural differentiation.
Bone and Bones ; CD146 Antigen/analysis* ; Cell Differentiation ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Humans ; Mesenchymal Stem Cells ; Neural Stem Cells ; Neurons ; Osteogenesis ; Staining and Labeling ; Tissue Engineering ; Tooth, Deciduous/cytology*

Mutations or inactivation of parkin, an E3 ubiquitin ligase, are associated with familial form or sporadic Parkinson's disease (PD), respectively, which manifested with the selective vulnerability of neuronal cells in substantia nigra (SN) and striatum (STR) regions. However, the underlying molecular mechanism linking parkin with the etiology of PD remains elusive. Here we report that p62, a critical regulator for protein quality control, inclusion body formation, selective autophagy and diverse signaling pathways, is a new substrate of parkin. P62 levels were increased in the SN and STR regions, but not in other brain regions in parkin knockout mice. Parkin directly interacts with and ubiquitinates p62 at the K13 to promote proteasomal degradation of p62 even in the absence of ATG5. Pathogenic mutations, knockdown of parkin or mutation of p62 at K13 prevented the degradation of p62. We further showed that parkin deficiency mice have pronounced loss of tyrosine hydroxylase positive neurons and have worse performance in motor test when treated with 6-hydroxydopamine hydrochloride in aged mice. These results suggest that, in addition to their critical role in regulating autophagy, p62 are subjected to parkin mediated proteasomal degradation and implicate that the dysregulation of parkin/p62 axis may involve in the selective vulnerability of neuronal cells during the onset of PD pathogenesis.
Adaptor Proteins, Signal Transducing ; chemistry ; metabolism ; Animals ; HEK293 Cells ; Heat-Shock Proteins ; chemistry ; metabolism ; Humans ; Lysine ; metabolism ; Mice ; Neurons ; metabolism ; pathology ; Oxidopamine ; pharmacology ; Parkinson Disease ; metabolism ; pathology ; Proteasome Endopeptidase Complex ; metabolism ; Protein Stability ; Proteolysis ; drug effects ; Sequestosome-1 Protein ; Ubiquitin-Protein Ligases ; metabolism ; Ubiquitination ; drug effects

OBJECTIVE: To compare the proliferation and capacity of differentiation to vascular endothelial cells and angiogenesis induction among stem cells from human exfoliated deciduous teeth (SHED), dental pulp stem cells (DPSC) and human bone marrow mesenchymal stem cells (BMSC) from orofacial bone. METHODS: SHED and DPSC were isolated from pulp tissue of the patients. BMSC were isolated from orthognathic or alveolar surgical sites. The surface markers of the cells were detected by flowcytometry. Cell counting kit-8 (CCK-8) assays were conducted to detect the proliferation ability of the cells. The cells were induced into endothelial cells with conditional medium and then the induced cells were cultured in Matrigel medium. The expression of angiogenesis-related genes such as platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrand Factor (vWF) were quantified by real-time PCR. The cells were cultured in chick embryo chorioallantoic membrane (CAM) and the vessels were counted after 5 days. RESULTS: The cell surface markers CD73, CD90, CD105 and CD146 of all the stem cells were positive, CD34 and CD45 were negative. The CD146 positive rate of SHED and DPSC was higher than that of BMSC. SHED had a higher proliferation rate than DPSC and BMSC. After angiogenic induction for 14 d, 3 kinds of cells emanated pseudopodia formed grid structure long vasculature in Matrigel media. The total length of tube formation of induced BMSC (7 759.7 μm) and SHED (7 734.3 μm) was higher than DPSC (5 541.0 μm). The meshes number of induced SHED (70.7) was higher than DPSC (60) and BMSC (53.7) in Matrigel medium. The expression of CD31, VEGFR2 and vWF genes of SHED were higher than those of BMSC and DPSC. VEGFR1 gene expression of BMSC was higher than that of the other groups, and SHED was higher than DPSC. The expression of VEGF showed no difference among the cells. No deference was showed between the effect of the stem cells and negative control on new formed vessels in CAM. The total length of vessels of SHED (30.4 mm) was higher than that of the negative control (20.9 mm) and BMSC (28.0 mm). CONCLUSION SHED, DPSC and BMSC can differentiate into vascular endothelial cells. SHED showed a stronger angiogenesis differentiation and proliferation potential compared with DPSC and BMSC.
Animals ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Chick Embryo ; Endothelial Cells ; Humans ; Mesenchymal Stem Cells ; Vascular Endothelial Growth Factor A