piggyBac Transposon-Based Immortalization of Human Deciduous Tooth Dental Pulp Cells with Multipotency and Non-Tumorigenic Potential

Autor: Masahiro Sato, Shinji Shibasaki, Youichi Yamasaki, Issei Saitoh, Naoko Kubota, Emi Inada, Yuki Kiyokawa, Yoko Iwase, Hirofumi Noguchi
Jazyk: angličtina
Rok vydání: 2019
Předmět:
0301 basic medicine
Transposable element
multi-differentiation potential
Biology
immortalization
piggybac
Catalysis
Article
Inorganic Chemistry
lcsh:Chemistry
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
Complementary DNA
Telomerase reverse transcriptase
Physical and Theoretical Chemistry
Molecular Biology
lcsh:QH301-705.5
Spectroscopy
Transposase
piggyBac
Organic Chemistry
telomerase reverse transcriptase
human deciduous tooth-derived dental pulp cell
General Medicine
Transfection
Molecular biology
Computer Science Applications
030104 developmental biology
stemness factor
chemistry
lcsh:Biology (General)
lcsh:QD1-999
Puromycin
Cell culture
030220 oncology & carcinogenesis
embryonic structures
tumorigenicity
Immortalised cell line
alkaline phosphatase
e7
Zdroj: International Journal of Molecular Sciences, Vol 20, Iss 19, p 4904 (2019)
International Journal of Molecular Sciences
Volume 20
Issue 19
ISSN: 1422-0067
Popis: We aimed to immortalize primarily isolated human deciduous tooth-derived dental pulp cells (HDDPCs) by transfection with piggyBac (PB)-based transposon vectors carrying E7 from human papilloma virus 16 or complementary DNA (cDNA) encoding human telomerase reverse transcriptase (hTERT). HDDPCs were co-transfected with pTrans (conferring PB transposase expression) + pT-pac (conferring puromycin acetyltransferase expression) + pT-tdTomato (conferring tdTomato cDNA expression) and pT-E7 (conferring E7 expression) or pTrans + pT-pac + pT-EGFP (conferring enhanced green fluorescent protein cDNA expression) + pT-hTERT (conferring hTERT expression). After six days, these cells were selected in medium containing 5 &mu
g/mL puromycin for one day, and then cultured in normal medium allowing cell survival. All resultant colonies were harvested and propagated as a pool. Stemness and tumorigenic properties of the established cell lines (&ldquo
MT_E7&rdquo
for E7 and &ldquo
MT_hTERT&rdquo
for hTERT) with untransfected parental cells (MT) were examined. Both lines exhibited proliferation similar to that of MT, with alkaline phosphatase activity and stemness-specific factor expression. They displayed differentiation potential into multi-lineage cells with no tumorigenic property. Overall, we successfully obtained HDDPC-derived immortalized cell lines using a PB-based transfection system. The resultant and parental cells were indistinguishable. Thus, E7 and hTERT could immortalize HDDPCs without causing cancer-associated changes or altering phenotypic properties.
Databáze: OpenAIRE
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