Telomerase-mediated life-span extension of human primary fibroblasts by human artificial chromosome (HAC) vector

https://doi.org/10.1016/j.bbrc.2008.02.119Get rights and content

Abstract

Telomerase-mediated life-span extension enables the expansion of normal cells without malignant transformation, and thus has been thought to be useful in cell therapies. Currently, integrating vectors including the retrovirus are used for human telomerase reverse transcriptase (hTERT)-mediated expansion of normal cells; however, the use of these vectors potentially causes unexpected insertional mutagenesis and/or activation of oncogenes. Here, we established normal human fibroblast (hPF) clones retaining non-integrating human artificial chromosome (HAC) vectors harboring the hTERT expression cassette. In hTERT-HAC/hPF clones, we observed the telomerase activity and the suppression of senescent-associated SA-β-galactosidase activity. Furthermore, the hTERT-HAC/hPF clones continued growing beyond 120 days after cloning, whereas the hPF clones retaining the silent hTERT-HAC senesced within 70 days. Thus, hTERT-HAC-mediated episomal expression of hTERT allows the extension of the life-span of human primary cells, implying that gene delivery by non-integrating HAC vectors can be used to control cellular proliferative capacity of primary cultured cells.

Section snippets

Materials and methods

Cells and culture conditions. The 21ΔqHAC vector was stably maintained in CHO-K1 cells cultured as previously described [12]. The HFL1 is a primary cultured fibroblast derived from human fetal lung (RCB0521, passage 1, RIKEN, Tsukuba, Japan). The HFL1 cells were maintained in DMEM containing 20% FBS, and were cultured in collagen-I-coated tissue culture flasks/plates (Falcon) in this study.

Fluorescence in situ hybridization. Preparation of metaphase chromosomes and hybridization were carried

Construction of the hTERT-HAC and Flpe-induced hTERT expression in CHO cells

To construct HACs carrying hTERT expression cassettes, we employed the hChr.21-derived, mini-chromosomal vector (21ΔqHAC) devoid of most part of the q-arm [2]. The 21ΔqHAC was stably maintained in the chinese hamster ovary (CHO) cell clone (KH21). The silent hTERT expression cassette, hTERT(OFF), is composed of the following DNA elements: a human ubiquitin C (UbC) promoter [26]; a FRT-flanked transcribe-translate termination sequence (STOP sequence) [27], [28]; a hTERT/internal ribosomal entry

Acknowledgments

We thank Drs. Makoto Kakitani and Ayano Inoue for valuable discussions.

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