Telomerase-mediated telomere elongation from human blastocysts to embryonic stem cells

Abstract

High telomerase activity is a characteristic of human embryonic stem cells (hESCs), however, the regulation and maintenance of correct telomere length in hESCs is unclear. In this study we investigated telomere elongation in hESCs in vitro and found that telomeres lengthened from their derivation in blastocysts through early expansion, but stabilized at later passages. We report that the core unit of telomerase, hTERT, was highly expressed in hESCs in blastocysts and throughout long-term culture; furthermore, this was regulated in a Wnt-b-catenin-signaling-dependent manner. Our observations that the alternative lengthening of telomeres (ALT) pathway was suppressed in hESCs and that hTERT knockdown partially inhibited telomere elongation, demonstrated that high telomerase activity was required for telomere elongation. We observed that chromatin modification through trimethylation of H3K9 and H4K20 at telomeric regions decreased during early culture. This was concurrent with telomere elongation, suggesting that epigenetic regulation of telomeric chromatin may influence telomerase function. By measuring telomere length in 96 hESC lines, we were able to establish that telomere length remained relatively stable at 12.0261.01 kb during later passages (15-95). In contrast, telomere length varied in hESCs with genomic instability and hESC-derived teratomas. In summary, we propose that correct, stable telomere length may serve as a potential biomarker for genetically stable hESCs.

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

“…28,35,47,48 However, telomere length is touted as a biomarker of reprogramming, with hiPSC telomere lengths plateauing close to the hESC telomere length of about 12 kbp despite continued high telomerase expression. 49 Such stabilization of telomere length, in addition to proper downregulation of hTERT following differentiation, is a strong hallmark of non-transformed human pluripotent stem cells. 30,35,49 hESCs and hiPSCs normally suppress the ALT (alternative lengthening of telomerase) pathway 49 and, in the absence of hTERT, short telomeres typically lead to up- regulation of the p53-mediated apoptosis pathway, or chromosomal rearrangements.…”

“…49 Such stabilization of telomere length, in addition to proper downregulation of hTERT following differentiation, is a strong hallmark of non-transformed human pluripotent stem cells. 30,35,49 hESCs and hiPSCs normally suppress the ALT (alternative lengthening of telomerase) pathway 49 and, in the absence of hTERT, short telomeres typically lead to up- regulation of the p53-mediated apoptosis pathway, or chromosomal rearrangements. 29 Thus, the aforementioned telomere maintenance characteristics in pluripotency are not preserved across vertebrates (Table 1).…”

“…60 Early lengthening of telomeres, within the first few passages following reprogramming, is preceded by a significant reduction of H3K9/H4K20 trimethylation. 49 However, this must be followed by reestablishment of H3K9/H4K20 me3 repressive marks to stabilize telomeric length. 49 While knockdown of histone methyltransferases (HMTs) SUV39h1 and SUV39h2 in mice and pigs leads to increased telomere length, decreased demethylases DNMT1/3a/3b and decreased H3K9me3 marks, 46 knockdown in human cells leads to telomere length shortening.…”

“…49 However, this must be followed by reestablishment of H3K9/H4K20 me3 repressive marks to stabilize telomeric length. 49 While knockdown of histone methyltransferases (HMTs) SUV39h1 and SUV39h2 in mice and pigs leads to increased telomere length, decreased demethylases DNMT1/3a/3b and decreased H3K9me3 marks, 46 knockdown in human cells leads to telomere length shortening. 46 This disparity is likely due to species differences ( Table 1) that repress ALT pathways in humans, but not murine or porcine cells.…”

“…33 Recently, KLF4 binding has been mapped directly to the TERT proximal promoter where KLF4 is able to upregulate TERT when b-catenin acts as a cofactor. 49,65,66 This appears to be a key function of KLF4, as hTERT overexpression is capable of rescuing KLF4 knockdown-triggered cellular differentiation. 66 Furthermore, KLF4-b-catenin in complex with TCF-4 or TCF-1 serves either to activate or to repress TERT, respectively.…”