Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation

P. Tátrai, Áron Szepesi, Zsolt Matula, Anna Szigeti, Gyöngyi Buchan, A. Mádi, F. Uher, K. Német

Research output: Contribution to journalArticle

27 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)28-35
Number of pages8
JournalBiochemical and Biophysical Research Communications
Volume422
Issue number1
DOIs
Publication statusPublished - May 25 2012

Fingerprint

Stromal Cells
Adipose Tissue
Tissue
Polyomavirus Transforming Antigens
Cells
Viral Tumor Antigens
human TERT protein
Karyotype
Cell Line
Phenotype
Regenerative Medicine
Cell Aging
Telomerase
Aberrations
Transgenes
Chromosome Aberrations

Keywords

  • Adipose tissue-derived stromal cells
  • HTERT
  • Immortalization

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation. / Tátrai, P.; Szepesi, Áron; Matula, Zsolt; Szigeti, Anna; Buchan, Gyöngyi; Mádi, A.; Uher, F.; Német, K.

In: Biochemical and Biophysical Research Communications, Vol. 422, No. 1, 25.05.2012, p. 28-35.

Research output: Contribution to journalArticle

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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.",
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AU - Uher, F.

AU - Német, K.

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