Telomere dynamics in human pluripotent stem cells

Abstract

Pluripotent stem cells (PSCs) are a promising source of stem cells for regenerative therapies. Stem cell function depends on telomere maintenance mechanisms that provide them with the proliferative capacity and genome stability necessary to multiply and regenerate tissues. We show here that established human embryonic stem cells (hESCs) have stable telomere length that is dependent on telomerase but not on alternative mechanisms based on homologous recombination pathways. Here, we show that human-induced pluripotent stem cells (hiPSCs) reprogrammed from somatic cells show progressive telomere lengthening until reaching a length similar to ESCs. hiPSCs also acquire telomeric chromatin marks of ESCs including decreased abundance of tri-methylated histone H3K9 and H4K20 and HP1 heterochromatic marks, as well as of the shelterin component TRF2. These chromatin features are accompanied with increased abundance of telomere transcripts or TERRAs. We also found that telomeres of both hESCs and hiPSCs are well protected from DNA damage during telomere elongation and once full telomere length is achieved, and exhibit stable genomes. Collectively, this study highlights that hiPSCs acquire ESC features during reprogramming and reveals the telomere biology in human pluripotent stem cells (hPSCs).

Keywords: Telomerase; Telomeres; hESCs; hiPSCs.

Figure 1.

BJ deficient for telomerase fail to be reprogrammed into iPSCs. a. Generation of TERT knock-out BJ fibroblasts by targeting the exon2 of TERT using the CRISPR/Cas9 technology. The sequences of BJ clones with successful TERT knockout are indicated. b. Generation of TERC knock-out BJ fibroblasts. Two sgRNAs flanking TERC were designed to delete TERC. Sequence of BJ clone with successful TERC deletion is indicated. Image of DNA electrophoresis gel showing the successful deletion of TERC is shown. c. Reprogramming of BJ fibroblasts with (WT) or without telomerase genes (TERT^−/−&TERC^−/−). Representative images of alkaline phosphatase (AP) staining showing iPS colonies are shown. Bright field images showing iPS colonies (yellow arrow) on feeders (top) and iPSCs established on feeder-free culture (bottom). d. Representative images of western blot analyzing NANOG, OCT4, TERT and TRF1 in different iPS clones at indicated passages by western blot. e. Protein level quantification. Bars represent mean values and error bars the standard deviation. One-way ANOVA was used for statistical analysis. n=number of independent clones.

Figure 2.

Progressive telomere elongation in reprogrammed hiPSCs.

Figure 3.

Telomerase is reactivated and maintained in hiPSCs.

Figure 4.

Telomeres of hiPSCs and hESCs are protected and genomically stable throughout successive passages.

Figure 5.

Telomeric chromatin is more “open” and telomeric RNA is upregulated in hiPSCs.

Figure 6.

hiPSCs with different telomere length show equal teratoma formation efficiency.