Telomere dynamics in human pluripotent stem cells

Figures & data

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.

a. Representative images of telomeric restriction fragment (TRF) blots of parental BJ fibroblasts and hiPS cell clones at different passages. Mean telomere length (Kb) is indicated at the bottom of each lane. Quantification of mean telomere length by TRF analysis of BJ and different iPS clones (C1-C6) at successive passages as indicated. Bars represent mean values and error bars the standard deviation. Paired t test was used for statistical analysis. n=number of independent clones. b. Representative microscopy Q-FISH images of metaphase spreads of different iPS clones (C1-C6) at passage 4 and 30. Quantification of mean telomere length by Q-FISH analysis of different iPS clones (C1-C6) in arbitrary units of fluorescence (a.u.f.) at successive passages as indicated. Bars represent mean values and error bars the standard deviation. Paired t test was used for statistical analysis. n=number of independent clones.

Figure 3. Telomerase is reactivated and maintained in hiPSCs.

a-b. Quantification TERT (a) and TERC (b) transcription levels by quantitative qPCR in BJ fibroblasts and in reprogrammed iPS clones at different passages. Bars represent mean values and error bars the standard deviation. Paired t test was used for statistical analysis. n=number of independent clones. c. Representative image of telomeric repeat amplification protocol (TRAP) for telomerase activity in ES, BJ and in iPSCs at different passages. The internal control (IC) was indicated and used for normalization. Quantification of the telomerase activity from the TRAP analysis. Bars represent mean values and error bars the standard deviation. Paired t test was used for statistical analysis. n=number of independent clones.

Figure 4. Telomeres of hiPSCs and hESCs are protected and genomically stable throughout successive passages.

a. Representative confocal microscopy images of telomere dysfunction induced foci (TIFs) in hiPSCs and hESCs at different passages. Co-localization of 53BP1 with telomere is recognized as a TIF. 6-thio-dG treated cells were used as a positive control. b-c. Quantification of the number of 53BP1 foci per nucleus (b) and percentage of cells with TIFs ≥ 2 (c). d. Representative microscopy images and quantification of multitelomeric signals (MTS) in hESCs and hiPSCs at different passages. Inset represents high magnification image. Bars represent mean values and error bars the standard deviation. One-way ANOVA was used for statistical analysis. n=number of independent clones.

Figure 5. Telomeric chromatin is more “open” and telomeric RNA is upregulated in hiPSCs.

a. Quantification of telomeric DNA pulled down with anti-H3K9me3, H4K20me3, HP1 and TRF2. ChIP values were normalized by the input of each individual sample. One-way ANOVA was used for statistical analysis. Bars represent mean values and error bars the standard deviation. n=number of independent experiments. Data from two different iPS clones were pooled together. b. Representative image of northern dot blot for TERRA in hES, BJ and hiPS clones 1 and 2 at different passages. Values were normalized against the signal for the 18S ribosomal subunit. One-way ANOVA was used for statistical analysis. Bars represent mean values and error bars the standard deviation. n=number of independent clones.

Figure 6. hiPSCs with different telomere length show equal teratoma formation efficiency.

a-b. Representative images of subcutaneous teratomas generated from hiPSCs with different telomere length at passage 4 (P4) and 30 (P30). Numbers indicate different mice. b-c. Teratoma volume follow-up (b) and teratoma weight measured at the human end point (c) in mice injected with hiPSCs at P4 and p30. Bars represent mean values and error bars the standard deviation. Paired t test was used for statistical analysis. n=number of mice. d. Representative images of hematoxylin & eosin staining of teratoma resulting from in vivo differentiation assay using hiPSCs at passage 4 and 30. Arrows mark the smooth muscle tissues. e-g. Representative immunohistochemistry images of Ki67 (e), P21 (f) and ɣ-H2Ax (g) expression in teratoma derived from hiPSCs at passages 4 and 30. Quantification plots are shown to the right. Paired t test was used for statistical analysis. Bars represent mean values and error bars the standard error. n=number of mice. h Representative image of telomeric Q-FISH in the neural epithelium cells of teratoma. Quantification of mean telomere length is shown to the right. a.u.f, arbitrary units of fluorescence. Bars represent mean values and error bars the standard error. Paired t test was used for statistical analysis. n=number of mice.

Data availability statement

All relevant data are within the manuscript and its Supporting Information files.