Establishment of ultra long‐lived cell lines by transfection of TERT into normal human fibroblast TIG‐1 and their characterization

Abstract

To establish useful human normal cell lines, TERT (telomerase reverse transcriptase) cDNA was transfected into normal female lung fibroblast, TIG-1. After long-term-sub-cultivation of 74 individual clones selected for resistance to G418, we obtained 55 cultures with normal range of life span [75 PDL (population doubling level)], 16 cultures with extended life span (75-140 PDL). In addition, 3 immortal cell strains and unexpectedly, one ultra long-lived cell line (ULT-1) with life span of 166 PDL were established. IMT-1, one of the immortal cell strains was confirmed to maintain long telomere length, high telomerase activity and an extremely low level of p16INK4A. They also showed moderate p53 and p21CIP1 expression, keeping vigorous growth rate even at 450 PDL. High level of fibronectin and collagen 1α expression confirmed IMT-1 as normal fibroblasts, although one X chromosome had been lost. ULT-1, however, kept a near normal karyotypes and had shortening of telomere length, high expression of p16INK4A, moderate levels of senescence associated-β-galactosidase positive cells and decreased growth rate only after 150 PDs (population doublings), and finally reached senescence at 166 PDL with morphology of normal senescent fibroblasts. As resources of standard normal human cell, abundant vials of early and middle passages of ULT-1 have been stocked. The use of the cell line is discussed, focusing on isograft of artificial skin and screening of anti-aging or safe chemical agents.

Changes of telomere status with aging: An update

“…Therefore, TERT is thought to be a gatekeeper of telomerase function. Forced expression of TERT in somatic cells can bypass replicative senescence . In contrast, reprograming of somatic cells by Yamanaka factors (induced pluripotent stem cells [iPS cells]) also activates TERT, and is associated with acquisition of self‐renewal capacity .…”

“…Forced expression of TERT in somatic cells can bypass replicative senescence. 85,86 In contrast, reprograming of somatic cells by Yamanaka factors (induced pluripotent stem cells [iPS cells]) also activates TERT, and is associated with acquisition of self-renewal capacity. 87,88 The telomere dynamics of iPS cells are one of the hottest focuses of telomere biology.…”

Reversible transformation and de-differentiation of human cells derived from induced pluripotent stem cell teratomas

“…Our recent study [ 10 ] on re-emergence of undifferentiated cells from hiPSC-derivatives prompted us to examine risks of transformation and de-differentiation of the differentiated cells. Further, a series of studies on cellular aging [ 11 ], immortalization [ 12 ], de-differentiation [ 10 ], and reprogramming [ 13 ] of human fibroblast TIG-1, urged us to isolate transformed cells from TIG-1 iPSC-teratomas and to disclose novel tumor risks of hiPSC-derivatives.…”

Reemergence of undifferentiated cells from transplants of human induced pluripotent stem cells is a possible potential risk factor of tumorigenic differentiation

“…We have previously generated iPSC lines from the normal human fibroblast TIG‐1 (Kumazaki et al, ) by using pCX‐OKS‐2A and pCX‐c‐Myc plasmids (Okita et al, ). And we have also established normal immortal cell lines from the same TIG‐1 (Kamada et al, ). These cell lines with the same genetic background are useful for the study of both aging and differentiation, and the former (iPSCs) are especially useful for the study of regenerative medicine.…”