piggyBac Transposon-Based Immortalization of Human Deciduous Tooth Dental Pulp Cells with Multipotency and Non-Tumorigenic Potential
Abstract
:1. Introduction
2. Results
2.1. Strategy for Generating Immortalized HDDPCs
2.2. Successful Establishment of Immortalized HDDPCs
2.3. Characterization of Established MT_E7 and MT_hTERT Lines
2.3.1. Transgenic Gene Expression and Cell Proliferation
2.3.2. Stemness
2.3.3. Multipotency
2.3.4. Immortalization
2.3.5. Tumorigenic potential
3. Discussion
4. Materials and Methods
4.1. Mice
4.2. Cells
4.3. Construction of PB Transposon Vectors
4.4. Generation of Immortalized HDDPC Lines
4.5. Fluorescence Observation
4.6. Cell Growth Assay
4.7. ALP Assay
4.8. PCR Analysis
4.9. RT-PCR Analysis
4.10. Immunocytochemical Staining
4.11. Induction of In Vitro Differentiation
4.12. Anchorage-Dependent Growth Assay
4.13. In Vivo Tumorigenic Assay
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
Abbreviations
HD | Human deciduous teeth |
DPCs | Dental pulp cells |
HDDPCs | Human deciduous teeth-derived dental pulp cells |
SV40 | Simian virus 40 |
HPV16 | Human papilloma virus 16 |
SV40 TAg | SV40 large T antigen |
HPV16-E7 | E7 oncoprotein of HPV16 |
hTR | Human telomerase RNA |
hTERT | Human telomerase reverse transcriptase |
hTEP1 | Human telomerase-associated protein 1 |
DPSCs | Dental pulp stem cells |
PB | piggyBac |
CAG | Chicken β-actin gene-based promoter |
pac | Puromycin acetyltransferase gene |
PGK | Phoshoglycerate kinase |
tdTomato | Tandem dimer Tomato |
EGFP | Enhanced green fluorescent protein |
GEU | Gene expression unit |
ALP | Alkaline phosphatase |
PBS | Dulbecco’s modified Ca2+, Mg2+-free phosphate-buffered saline |
iPS | Induced pluripotent stem |
OCT-3/4 | Octamer-binding transcription factor-3/4 |
SOX2 | Sex determining region Y-box 2 |
DSPP | Dentin sialophosphoprotein |
IP | Intraperitoneal |
DMEM | Dulbecco’s modified Eagle’s medium |
FBS | Fetal bovine serum |
PFA | Paraformaldehyde |
RT | Room temperature |
PBS | Dulbecco’s modified Ca2+, Mg2+-free phosphate-buffered saline |
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase |
NGS | Normal goat serum |
DAPI | 4′,6-diamidino-2-phenylindole |
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Inada, E.; Saitoh, I.; Kubota, N.; Iwase, Y.; Kiyokawa, Y.; Shibasaki, S.; Noguchi, H.; Yamasaki, Y.; Sato, M. piggyBac Transposon-Based Immortalization of Human Deciduous Tooth Dental Pulp Cells with Multipotency and Non-Tumorigenic Potential. Int. J. Mol. Sci. 2019, 20, 4904. https://doi.org/10.3390/ijms20194904
Inada E, Saitoh I, Kubota N, Iwase Y, Kiyokawa Y, Shibasaki S, Noguchi H, Yamasaki Y, Sato M. piggyBac Transposon-Based Immortalization of Human Deciduous Tooth Dental Pulp Cells with Multipotency and Non-Tumorigenic Potential. International Journal of Molecular Sciences. 2019; 20(19):4904. https://doi.org/10.3390/ijms20194904
Chicago/Turabian StyleInada, Emi, Issei Saitoh, Naoko Kubota, Yoko Iwase, Yuki Kiyokawa, Shinji Shibasaki, Hirofumi Noguchi, Youichi Yamasaki, and Masahiro Sato. 2019. "piggyBac Transposon-Based Immortalization of Human Deciduous Tooth Dental Pulp Cells with Multipotency and Non-Tumorigenic Potential" International Journal of Molecular Sciences 20, no. 19: 4904. https://doi.org/10.3390/ijms20194904