title: Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation.
abstract: Adipose tissue-derived stromal cells (ASCs) are increasingly being studied for their usefulness in regenerative medicine. However, limited life span and donor-dependent variation of primary cells such as ASCs present major hurdles to controlled and reproducible experiments. We therefore aimed to establish immortalized ASC cell lines that provide steady supply of homogeneous cells for in vitro work while retain essential features of primary cells. To this end, combinations of human telomerase reverse transcriptase (hTERT), murine Bmi-1, and SV40 large T antigen (SV40T) were introduced by lentiviral transduction into ASCs. The resulting cell lines ASC(hTERT), ASC(Bmi-1), ASC(Bmi-1+hTERT) and ASC(SV40T+hTERT) were tested for transgene expression, telomerase activity, surface immunomarkers, proliferation, osteogenic and adipogenic differentiation, karyotype, tumorigenicity, and cellular senescence. All cell lines have maintained expression of characteristic surface immunomarkers, and none was tumorigenic. However, ASC(Bmi-1) had limited replicative potential, while the rapidly proliferating ASC(SV40T+hTERT) acquired chromosomal aberrations, departed from MSC phenotype, and lost differentiation capacity. ASC(hTERT) and ASC(hTERT+Bmi-1), on the other hand, preserved all essential MSC features and did not senesce after 100 population doublings. Notably, a subpopulation of ASC(hTERT) also acquired aberrant karyotype and showed signs of transformation after long-term culture. In conclusion, hTERT alone was sufficient to extend the life span of human ASC, but ASC(hTERT) are prone to transformation during extensive subculturing. The combination of Bmi-1 and hTERT successfully immortalized human ASCs without significantly perturbing their phenotype or biological behavior.
authors.affiliation: Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, Karolina út 29, H-1113 Budapest, Hungary. peter.tatrai@biomembrane.hu
authors.forename: Péter
authors.initials: P
authors.lastname: Tátrai
authors.forename: Áron
authors.initials: Á
authors.lastname: Szepesi
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Matula
authors.forename: Anna
authors.initials: A
authors.lastname: Szigeti
authors.forename: Gyöngyi
authors.initials: G
authors.lastname: Buchan
authors.forename: András
authors.initials: A
authors.lastname: Mádi
authors.forename: Ferenc
authors.initials: F
authors.lastname: Uher
authors.forename: Katalin
authors.initials: K
authors.lastname: Német
mesh_terms: D000273:Adipose Tissue / Q000166:cytology / Q000473:pathology / Q000502:physiology*; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D016922:Cellular Senescence / Q000235:genetics / Q000502:physiology*; D018014:Gene Transfer Techniques; D006801:Humans; D059785:Karyotype; D016086:Lentivirus; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics*; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D017154:Stromal Cells / Q000166:cytology / Q000473:pathology / Q000502:physiology; D019098:Telomerase / Q000235:genetics*
chemical_list: C489421:Bmi1 protein, mouse; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D063150:Polycomb Repressive Complex 1; C509186:TERT protein, human; D019098:Telomerase