title: PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells.
abstract: The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine.
authors.affiliation: Department of Pediatric Dentistry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.
authors.forename: Emi
authors.initials: E
authors.lastname: Inada
authors.affiliation: Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan.
authors.forename: Issei
authors.initials: I
authors.lastname: Saitoh
authors.affiliation: Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ibaraki, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Functional Biomolecules Research Group, National Agriculture and Food Research Organization, Ibaraki, Japan.
authors.forename: Reiji
authors.initials: R
authors.lastname: Aoki
authors.affiliation: Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Miura
authors.affiliation: Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Ohtsuka
authors.affiliation: Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Murakami
authors.affiliation: Division of Pediatric Dentistry, Graduate School of Medical and Dental Science, Niigata University, Niigata, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Sawami
authors.affiliation: Department of Pediatric Dentistry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.
authors.forename: Youichi
authors.initials: Y
authors.lastname: Yamasaki
authors.affiliation: Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
mesh_terms: D002478:Cells, Cultured; D004251:DNA Transposable Elements; D003782:Dental Pulp / Q000166:cytology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D014094:Tooth, Deciduous / Q000166:cytology*; D014162:Transfection
chemical_list: D004251:DNA Transposable Elements; D009419:Nerve Tissue Proteins; C569369:PGBD1 protein, human