ABSTRACT
Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods.In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.
ABSTRACT
Direct reprogramming, also known as a trans-differentiation, is a technique to allow mature cells to be converted into other types of cells without inducing a pluripotent stage. It has been suggested as a major strategy to acquire the desired type of cells in cell-based therapies to repair damaged tissues. Studies related to switching the fate of cells through epigenetic modification have been progressing and they can bypass safety issues raised by the virus-based transfection methods.In this study, a protocol was established to directly convert fully differentiated fibroblasts into diverse mesenchymal-lineage cells, such as osteoblasts, adipocytes, chondrocytes, and ectodermal cells, including neurons, by means of DNA demethylation, immediately followed by culturing in various differentiating media. First, 24 h exposure of 5-azacytidine (5-aza-CN), a well-characterized DNA methyl transferase inhibitor, to NIH-3T3 murine fibroblast cells induced the expression of stem-cell markers, that is, increasing cell plasticity. Next, 5-aza-CN treated fibroblasts were cultured in osteogenic, adipogenic, chondrogenic, and neurogenic media with or without bone morphogenetic protein 2 for a designated period. Differentiation of each desired type of cell was verified by quantitative reverse transcriptase-polymerase chain reaction/western blot assays for appropriate marker expression and by various staining methods, such as alkaline phosphatase/alizarin red S/oil red O/alcian blue. These proposed procedures allowed easier acquisition of the desired cells without any transgenic modification, using direct reprogramming technology, and thus may help make it more available in the clinical fields of regenerative medicine.
ABSTRACT
BACKGROUND@#Molar incisor hypomineralization (MIH) frequently occurs in children worldwide. However, MIH prevalence throughout Japan has not yet been investigated. The purpose of this study was to clarify MIH prevalence rates and to consider potential regional differences throughout Japan.@*METHODS@#A total of 4496 children aged 7-9 years throughout Japan were evaluated in this study. MIH prevalence rates among children were evaluated in eight regions throughout Japan. A child's residence was defined as the mother's residence during pregnancy. The localization of demarcated opacities and enamel breakdown was recorded on a standard code form using a guided record chart. Logistic regression analysis was used to evaluate whether MIH prevalence rates differed among age groups, sex, and regions.@*RESULTS@#The overall prevalence of MIH in Japan was 19.8%. The prevalence of MIH was 14.0% in the Hokkaido region, 11.7% in the Tohoku region, 18.5% in the Kanto Shin-Etsu region, 19.3% in the Tokai Hokuriku region, 22.3% in the Kinki region, 19.8% in the Chugoku region, 28.1% in the Shikoku region, and 25.3% in the Kyushu region. These regional differences were statistically significant. Moreover, MIH prevalence rates decreased with age. No significant sex differences in MIH prevalence rates were demonstrated.@*CONCLUSIONS@#To our knowledge, this is the first MIH study carried out in several regions throughout Japan. Regional differences existed in MIH prevalence rates; particularly, MIH occurred more frequently in children residing in southwestern areas than those in northeastern areas of Japan.