ABSTRACT
OBJECTIVE: The aim of our study was to investigate the effect of Transforming growth factor beta-1 (TGF-β1) gene therapy on the surface markers, multilineage differentiation, viability, apoptosis, cell cycle, DNA damage and senescence of human Dental Pulp-derived Mesenchymal Stromal Cells (hDPSC). METHODS: hDPSCs were isolated from human teeth, and were cultured with 20% Fetal Bovine Serum (FBS) in minimum essential media-alpha (α-MEM). TGF-β1 gene transfer into hDPSCs was performed by electroporation method after the plasmid was prepared. The transfection efficiency was achieved by using western blot and flow cytometry analyses and GFP transfection. Mesenchymal stem cell (MSC) markers, multilineage differentiation, cell proliferation, apoptosis, cell cycle, DNA damage and cellular senescence assays were performed by comparing the transfected and non-transfected cells. Statistical analyses were performed using GraphPad Prism. RESULTS: Strong expression of TGF-β1 in pCMV-TGF-β1-transfected hDPSCs was detected in flow cytometry analysis. TGF-β1 transfection efficiency was measured as 95%. Western blot analysis showed that TGF-β1 protein levels increased at third and sixth days in pCMV-TGF-β1-transfected hDPSCs. The continuous TGF-β1 overexpression in hDPSCs did not influence the immunophenotype and surface marker expression of MSCs. Our results showed that TGF-β1 increased osteogenic and chondrogenic differentiation, but decreased adipogenic differentiation. Overexpression of TGF-β1 increased the proliferation rate and decreased total apoptosis in hDPSCs (p<0.05). The number of cells at “S” phase was higher with TGF-β1 transfection (p<0.05). Cellular senescence decreased in TGF-β1 transfected group (p<0.05). CONCLUSIONS: These results reflect that TGF-β1 has major impact on MSC differentiation. TGF-β1 transfection has positive effect on proliferation, cell cycle, and prevents cellular senescence and apoptosis.
Subject(s)
Humans , Aging , Apoptosis , Blotting, Western , Cellular Senescence , Cell Cycle , Cell Differentiation , Cell Proliferation , DNA Damage , Electroporation , Flow Cytometry , Genetic Therapy , Mesenchymal Stem Cells , Methods , Plasmids , Population Characteristics , Tooth , Transfection , Transforming Growth FactorsABSTRACT
Objective:To culture and characterize the deciduous tooth pulp stem cells(DTPSCs)of Beagle dog.Methods:DTPSCs were cultured using enzyme tissue block method from Beagle dog aged 6 weeks.The cells were purified by cloning culture,identified by immunohistochemistry and flowcytometry.The biological characteristics were studied by CCK-8 assay,osteogenetic induction,li-pogenic induction and dentinogenic induction assays.Results:Beagle stem cells from deciduous tooth pulp were obtained,the cell colony formation rate was 32%.The cells were STRO-1 and CD146 positive,CD14,CD45 and CD86 negative.After multiple induc-tion culture the cells were positive for alizarin red staining,oil red staining,ALP expression and DSSPP expression.Conclusion:The deciduous tooth pulp stem cells of Beagle dog have multilineage differentiation abilities.
ABSTRACT
Recientemente se ha descubierto que diversos tejidos dentales son fuente importante de Células Madre Mesenquimales (CMM). En la cavidad oral podemos encontrar CMM en la pulpa, en el folículo dental, papila y en la encía entre otros lugares. Varios estudios avalan el extenso potencial terapéutico de las CMM en terapias de regeneración. El objetivo de este estudio es aislar, cultivar células madres mesenquimales de pulpa y folículo dental humano, caracterizar su inmunofenotipo y su potencial de diferenciación a linaje osteogénico, condrogénico y osteogénico. Se cultivaron células de pulpa y folículo dental de terceros molares de dientes permanentes jóvenes humanos. Los cultivos de CMM fueron monitoreados por microscopia óptica, las células se inmunotipificaron por citometría de flujo. Posteriormente se evaluó su capacidad de diferenciaron a los tres linajes mencionados. En estas condiciones experimentales se comprobó que las células aisladas y cultivadas de pulpa y folículo dental correspondían a células madre mesenquimales humanas, siendo éstas últimas más fáciles de obtener y proliferar. Las CMM de folículo dental poseen mayor potencial de crecimiento y capacidad de diferenciación en comparación a las CMM de pulpa dental, probablemente debido a su estado evolutivo más inmaduro.
It was recently discovered that dental tissues are important sources of mesenchymal stem cells (MSCs). In the oral cavity MSCs can be found in the pulp, dental follicle, apical papilla and gingival tissue, among others. Many studies support the therapeutic potential of MSCs in regenerative therapies. The objective of this study was to isolate and culture mesenchymal stem cells from human dental pulp and follicle, and to characterize their immunophenotype and differentiation potential to adipogenic, chondrogenic and osteogenic lineages. Oral cavity stem cells were cultured from pulp and dental follicle of wisdom teeth from young permanent teeth. Immunotypification of MSCs was performed by flow cytometry and cultures were evaluated for their ability to differentiate into the three lineages mentioned. Our results corroborate that cultured oral MSC cells isolated from pulp and dental follicle were mesenchymal in origin, being the latter more easy to obtain. Dental follicle MSCs have greater growth potential and differentiation capacity compared to dental pulp MSCs, probably due to their more immature developmental state.