Robust activation of the human but not mouse telomerase gene during the induction of pluripotency

Abstract

Pluripotent stem cells (PSCs) express telomerase and have unlimited proliferative potential. To study telomerase activation during reprogramming, 3 classes of embryonic stem cell (ESC)-like clones were isolated from mouse fibroblasts containing a transgenic hTERT reporter. Class I expressed few pluripotency markers, whereas class II contained many, but not Oct4, Nanog, and Sox2. Neither class of cells differentiated efficiently. Class III cells, the fully reprogrammed induced PSCs (iPSCs), expressed all pluripotency markers, formed teratomas indistinguishable from those of mESCs, and underwent efficient osteogenic differentiation in vitro. Interestingly, whereas the endogenous mTERT gene expression was only moderately increased during reprogramming, the hTERT promoter was strongly activated in class II cells and was further elevated in class III cells. Treatment of class II cells with chemical inhibitors of MEKs and glycogen synthase kinase 3 resulted in their further reprogramming into class III cells, accompanied by a strong activation of hTERT promoter. In reprogrammed human cells, the endogenous telomerase level, although variable among different clones, was dramatically elevated. Only in cells with the highest telomerase were telomeres restored to the lengths in hESCs. Our data, for the first time, demonstrated that the hTERT promoter was strongly activated in discrete steps, revealing a critical difference in human and mouse cell reprogramming. Because telomere elongation is crucial for self-renewal of hPSCs and replicative aging of their differentiated progeny, these findings have important implications in the generation and applications of iPSCs.

The role of telomeres and telomerase reverse transcriptase isoforms in pluripotency induction and maintenance

“…27,28 Similarly, human and murine induced pluripotent stem cells (iPSCs) are capable of carrying out re-elongation of telomeres in at least some clones, although the telomere length and activity characteristics appear to be clonally unique. [28][29][30][31][32][33] Conversely, the process of differentiation triggers down-regulation of telomerase and initiates telomeric attrition. 34,35 Following the extended cellular division required to form adult tissues, terminally differentiated cell types typically exhibit low or undetectable levels of telomerase activity and shortened telomeres.…”

“…31,33,46 Partially reprogrammed iPSCs often show little TERT activation and short telomeres. 32 This reprogramming failure is likely the reason for the failure of the TERT promoter to be activated. Conversely, it is unlikely that lack of TERT activation causes partial reprogramming as TERT expression and/or activity does not guarantee pluripotency.…”

“…52 While NT-mESCs generally have completed telomere lengthening and full reprogramming by the blastocyst stage, 44 miPSCs may take up to 30 passages to achieve mESC lengths 53 and only do so sporadically. 32 Control of telomere length is an intricate process involving many factors. As previously reviewed, 19 mESCs and miPSCs control telomere length largely by modulating telomeric epigenetics and regulation by several proteins, including ZSCAN4, 51,[54][55][56] ATRX, 54,55 RIF1, 56 TRF1 52,57 TPP1 and other shelterin complex components.…”

Telomere dynamics in induced pluripotent stem cells: Potentials for Human disease modeling

“…It was demonstrated that iPSCs could not be generated from somatic cells from late generations (G3) of telomerase-null mice, possibly due to the high degrees of genomic instability as a result of telomere fusions, and that reintroduction of telomerase could restore the efficiency of generating iPSCs [50] . Interestingly, iPSCs derived from normal human and mouse cells show progressive telomere elongation with passaging in cultures, indicating that telomere elongation can occur postprogramming [50][51][52][53] . Studies have also shown that telomerase enzymatic activity is significantly activated upon iPS manipulation [38][39][40]50,52,54] as a result of upregulated expression of the TERT protein and TERC RNA, as well as of the associated protein dyskerin [52,54] .…”

“…Interestingly, iPSCs derived from normal human and mouse cells show progressive telomere elongation with passaging in cultures, indicating that telomere elongation can occur postprogramming [50][51][52][53] . Studies have also shown that telomerase enzymatic activity is significantly activated upon iPS manipulation [38][39][40]50,52,54] as a result of upregulated expression of the TERT protein and TERC RNA, as well as of the associated protein dyskerin [52,54] . The level of increase in TERT transcript and telomerase function, however, differs between human and mouse cells, about 100 fold in human iPSCs and a modest 2-3 fold in mouse iPSCs, possibly reflecting mechanistic differences in telomerase regulation in different organisms.…”