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.

Introduction

Mesenchymal stem cells (MSCs) are adult multipotent cells with immense therapeutic potential. MSCs were first discovered in the bone marrow stroma, but now it is widely accepted that cells with similar properties can be isolated from a variety of tissues [1]. While most experiments and clinical studies still focus on bone marrow-derived MSCs, adipose tissue is emerging as a promising alternative source of MSCs for the ease and relatively small discomfort of harvesting, and the high density of stem cells, specifically termed adipose-derived stromal cells or ASCs, in fat tissue [2].

Although the main therapeutic features of MSCs such as multilineage differentiation, regeneration-promoting trophic effect, and immunomodulation have been subject to intensive research in the last two decades, many of the underlying mechanisms remain to be clarified. Yet, controlled and reproducible in vitro work with MSC-like cell types is hampered by variation between cells isolated from different individuals and limited life span of primary cultures. Bone marrow MSCs from healthy donors exhibit striking heterogeneity in terms of both growth and differentiation, and the differences observed show no obvious correlation with age or gender [3]. Repeated use of cells from a single donor could eliminate inter-individual variation, but MSCs gradually lose multipotency and eventually become senescent as they undergo extensive proliferation ex vivo [4].

Both heterogeneity and replicative senescence can be circumvented by the establishment of immortal cell lines from primary cell cultures. Since cellular senescence is associated with telomere shortening, immortalization strategies typically include forced expression of telomerase reverse transcriptase (hTERT). Successful immortalization of bone marrow MSCs from human, minipig, and rhesus monkey has been achieved by expressing exogenous hTERT [5], [6], [7], [8]. However, several investigators have found that restoring telomerase activity is not sufficient for the immortalization of human primary cells [9], [10]. Therefore, in other experiments hTERT was introduced into human bone marrow- or placenta-derived MSCs in combination with growth promoters such as human papillomavirus (HPV) E6/E7 oncoprotein or the p16^Ink4a antagonist Bmi-1 [11], [12]. In some instances, hTERT indeed proved to be insufficient for immortalization; moreover, observations made on bone marrow MSCs transduced with HPV E6/E7 [13] and embryonic stem cell-derived MSCs transduced with c-MYC [14] indicated that hTERT may even be dispensable under certain conditions.

Immortalization of ASCs of either human or animal origin has not been reported to date. Hence we set out to establish immortalized human ASC cell lines that retain essential features of primary cells including differentiation potential and growth capacity over large time scales, and thus provide steady supply of homogeneous cells for in vitro work. We used the lentiviral technique to introduce hTERT and murine Bmi-1, both separately and in combination, into human primary ASCs. For positive biological control of immortalization, hTERT was combined with SV40T, the prototypical and most potent immortalizing gene [15].