Prevention of senescence progression in reversibly immortalized human ensheathing glia permits their survival after deimmortalization.
Reversible immortalization holds great potential for primary tissue expansion to develop cell-based therapies as well as for basic research. Human olfactory ensheathing glia (hOEG) are promising candidates for treating spinal cord injury and for studying extrinsic neuroregenerative mechanisms. We used lentivectors with Cre/loxP technology to achieve reversible gene transfer of BMI1, SV40 large T antigen (TAg), a short hairpin RNA against p53 (shp53), and the catalytic subunit of telomerase (TERT) in primary cultures of hOEG from human donor cadaver olfactory bulbs. Several combinations of these genes were able to immortalize hOEG, conserving their antigenic markers and neuroregenerative properties but only those transduced by BMI1/TERT did not accumulate karyotypic alterations or increase senescence marker levels. Strikingly, these were also the only cells which continued to proliferate after transgene removal by Cre recombinase delivery, whereas hOEG immortalized by shp53 or TAg in combination with TERT entered into growth arrest and died. These data support the idea that immortalization and halting senescent changes are separate processes; hOEG immortalized by BMI1/TERT can revert back to their former primary cell replicative state when deimmortalized, whereas those transduced by the other combinations depend on the presence of these transgenes to maintain their aberrant proliferative state.
García-Escudero
Vega
V
Departamento de Biología Molecular, Universidad Autónoma de Madrid, Madrid, Spain.
García-Gómez
Ana
A
Gargini
Ricardo
R
Martín-Bermejo
María J
MJ
Langa
Elena
E
de Yébenes
Justo G
JG
Delicado
Alicia
A
Avila
Jesús
J
Moreno-Flores
María T
MT
Lim
Filip
F
D000293:Adolescent; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000235:genetics / Q000502:physiology*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D007621:Karyotyping; D016086:Lentivirus / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D009830:Olfactory Bulb / Q000166:cytology*; D063150:Polycomb Repressive Complex 1; D011518:Proto-Oncogene Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019098:Telomerase / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D000952:Antigens, Polyomavirus Transforming; C069077:BMI1 protein, human; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53; D063150:Polycomb Repressive Complex 1; D019098:Telomerase