Dynamics of Telomere Rejuvenation during Chemical Induction to Pluripotent Stem Cells

Abstract

Chemically induced pluripotent stem cells (CiPSCs) may provide an alternative and attractive source for stem cell-based therapy. Sufficient telomere lengths are critical for unlimited self-renewal and genomic stability of pluripotent stem cells. Dynamics and mechanisms of telomere reprogramming of CiPSCs remain elusive. We show that CiPSCs acquire telomere lengthening with increasing passages after clonal formation. Both telomerase activity and recombination-based mechanisms are involved in the telomere elongation. Telomere lengths strongly indicate the degree of reprogramming, pluripotency, and differentiation capacity of CiPSCs. Nevertheless, telomere damage and shortening occur at a late stage of lengthy induction, limiting CiPSC formation. We find that histone crotonylation induced by crotonic acid can activate two-cell genes, including Zscan4; maintain telomeres; and promote CiPSC generation. Crotonylation decreases the abundance of heterochromatic H3K9me3 and HP1α at subtelomeres and Zscan4 loci. Taken together, telomere rejuvenation links to reprogramming and pluripotency of CiPSCs. Crotonylation facilitates telomere maintenance and enhances chemically induced reprogramming to pluripotency.

Keywords: Zscan4; chemically induced pluripotent stem cells; crotonic acid; genome stability; telomeres.

Graphical abstract

Figure 1

Pluripotency and Differentiation Capacity of CiPSCs (A) Representative morphology of CiPSCs generated by two methods (BrdU method, CiPSC1b at P12, and three-step method, CiPSC2t at P10) under bright field (BF) with phase contrast optics and expression of Oct4-GFP fluorescence. Scale bar represents 100 μm. (B) Immunofluorescence microscopy of pluripotent markers SOX2, NANOG, and SSEA1. Scale bar represents 10 μm. (C) mRNA expression levels by qPCR of Oct4, Nanog, and Rex1 in CiPSCs at various passages, compared with isogenic ESCs (OG4) and progenitor MEFs. Data represent mean ± SEM from three independent experiments. (D) Protein levels of OCT4, NANOG, and SOX2 by western blot analysis of CiPSCs at earlier and advanced passages. (E) Differentiation capacity in vitro of CiPSCs by immunofluorescence microscopy of three germ layer markers. Scale bar represents 10 μm. (F) Left photo represents chimeras generated from the BrdU method and the right from the three-step method. (G) Summary table showing percentage of chimeras generated from CiPSCs at different passages compared with OG4 ESCs. Chimeras (black and albino coat) were initially identified by coat color and some confirmed by microsatellite genotyping. CiPSC1b and 7b were generated using the BrdU method and CiPSC2t and the 6t by three-step method. See also Figure S1.

Figure 2

Telomere Rejuvenates in CiPSCs with Passages (A) Telomerase activity by telomeric repeat amplification protocol (TRAP) assay of CiPSCs during passages compared with progenitor cell MEFs. Lysis buffer served as negative control and OG4 ESCs as positive control. (B and C) Telomere length distribution shown as TRF by Southern blot analysis of MEFs and CiPSCs following passages. (D) Representative images displaying telomere FISH of MEF and CiPSCs at various passages. Blue, chromosomes stained with DAPI; green dots, telomeres. (E) Line progression plot showing dynamics of relative telomere length by Q-FISH shown as telomere fluorescence unit (TFU) of MEFs and CiPSCs for each cell line from early to late passage. Left, BrdU method; right, three-step method. (F) Scatterplots showing comparison of genome-wide transcription profile between CiPS1b or CiPS2t and ESC (OG4) or MEF. Parallel diagonal lines indicate 2-fold threshold in expression difference. Red, upregulated genes; green, downregulated genes. (G) ZSCAN4 protein levels by western blot analysis. MEF and ESCs (OG4) served as negative and positive controls, respectively. β-ACTIN served as loading control. (H) C-circle assay. Hela and U2OS served as negative and positive controls, respectively. (I) Representative micrographs showing telomere sister chromatid exchange (T-SCE, white arrows) by chromosome orientation FISH analysis. (J) Frequency of T-SCE. n, number of spread counted. ^∗∗∗p < 0.001, compared with MEFs. Two independent experiments. See also Figures S2 and S3.

Figure 3

Telomere Length Indicates Differentiation Capacity by Teratoma Formation (A) Differentiation in vivo of CiPSCs at early and late passages by teratoma formation test. (B) Weight of teratomas formed from CiPSCs, Mean ± SEM (n = 6 mice). ^∗p < 0.05, ^∗∗p < 0.01. (C) Linear regression analysis showing high correlation between telomere length (TFU by Q-FISH) and teratomas weight of CiPSCs. (D) Histology by H&E staining of teratoma tissues derived from early and late passage CiPSCs. Scale bar represents 50 μm. (E) Immunofluorescence of the teratomas showing markers representative of three germ layers: NESTIN (ectoderm), SMA (mesoderm), and AFP (endoderm). Scale bar represents 100 μm.

Figure 4

Telomere Dynamics during CiPSC Induction (A) Morphological changes of MEFs during chemical induction under bright field with phase contrast optics or Oct4-GFP fluorescence. Compact colonies were formed at day 40 of induction by three-step method, earlier than with the BrdU method. Scale bar represents 100 μm. (B) mRNA expression levels of Oct4, Sox2, Nanog, and Zscan4 during chemical induction. (C) Relative expression levels of Tert and Terc during chemical induction. (D) Telomerase activity by TRAP assay of MEFs and reprogramming cells during induction, compared with CiPSCs at P4 or P5. Lysis buffer as negative control. (E) Telomere length distribution shown as TRF by Southern blot analysis during chemical induction (day 0–40) compared with CiPSCs. MW, molecular weight. (F) Immunofluorescence showing co-staining of γH2AX (green) at telomeres (TRF1, red). Yellow (white arrows) in merged images indicates co-localized foci of TRF1 and γH2AX. Scale bar represents 5 μm. (G) Quantification of TIFs. n = 100 cells counted. Data represent mean ± SEM from three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. See also Figure S4.

Figure 5

Crotonic Acid Addition at Stage II Activates Zscan4, Maintains Telomeres, and Promotes CiPSC Generation (A) Durations of crotonic acid (7 mM) addition at different time points during chemical induction and assessment of Oct4-GFP-positive colonies on day 40. Left panel shows different time points during reprogramming. Stage I (SI) represents days 0–15; stage II (SII) represents days 16–28; stage III (SIII) represents days 29–40. Right panel shows number of Oct4-GFP-positive colonies on day 40; 100,000 cells were re-plated on day 12 following induction. ^∗∗∗p < 0.001, compared with control. (B) Morphology by phase contrast optics and Oct4-GFP fluorescence of primary CiPSC colonies on day 40 induced with or without crotonic acid (CA) at stage II. Scale bar represents 100 μm. (C) Telomerase activity by TRAP assay. Lysis buffer and ESCs served as negative and positive controls, respectively. (D) Immunofluorescence microscopy of ZSCAN4 and Kcr (lysine crotonylation) expression. Scale bar represents 10 μm. (E) Percentage of Zscan4-positive cells. Number of cells counted is shown above the bar. (F) Western blot analysis of protein levels. MEF and CiPSCs served as controls. (G) Telomere length distribution shown as TRF by Southern blot analysis. (H) Quantification of telomere length by TRF in three repeats using TeloTool (Gohring et al., 2014). ^∗p < 0.05. (I) Immunofluorescence of γH2AX (green) at telomeres (TRF1, red). Yellow foci in the merged images indicate TRF1-γH2AX co-localization. Scale bar represents 5 μm. Bottom panel, quantification of TIFs. n = 100 cells counted. ^∗∗p < 0.01, n.s., not significant. Data represent mean ± SEM from three independent experiments. See also Figure S5.

Figure 6

RNA-Seq Reveals Crotonic Acid-Induced Activation of 2C Genes (A) Scatterplots showing global differential gene expression profile (fold ≥ 2.0) of reprogramming cells on days 20–28 following treatment with CA in comparison with controls. Red, upregulated genes; green, downregulated genes in CA-treated cells. Average from two independent experiments. (B) Gene ontology analysis of differentially expressed genes in cells treated with crotonic acid, compared with controls. (C) Fisher's exact test between relatively upregulated differential genes at early embryo developmental stages from single-cell RNA-seq data (Fan et al., 2015) and all 196 differential genes from our RNA-seq data (p adjusted < 0.01). (D) Heatmap highlighting highly expressed 2C genes found in CA-treated cells compared with controls. (E) ChIP-qPCR analysis of H3K9me3 and HP1α abundance at Zscan4, Chr13_subtel (sub-telomere region of no. 13 chromosome), and ERVK10c loci following treatment with crotonic acid for 12 days during stage II of induction. β-actin served as negative control. Data represent mean ± SD from three independent experiments. ^∗p < 0.05. (F) Schematic diagram illustrating the process of CiPSC induction following addition of crotonic acid, which activates 2C genes including Zscan4, reduces telomere and DNA damage, and maintains telomeres, promoting CiPSC formation. See also Figure S6.

Figure 7

Generation and Characterization of CaCiPSCs Induced by Addition of Crotonic Acid at Stage II (A) Morphology under bright field with phase contrast optics and Oct4-GFP fluorescence of CiPSCs generated without CA and CaCiPSCs with CA. Scale bar represents 100 μm. (B) Scatterplots comparing transcriptome profile of CaCiPS1t from CA-treated cells, CiPS2t without CA treatment, OG4 ESCs, and MEFs. Parallel diagonal lines indicate 2-fold threshold. (C) Hierarchical clustering of transcriptome profile of MEFs, OG4 ESCs, CiPS1b, CiPS2t, CaCiPS1t, and CaCiPS4t. (D and E) Summary table showing efficiency of chimera generation from CaCiPSCs at mid and late passages following injection into four- to eight-cell albino embryos (D). Chimeras were initially identified by coat color (E) and confirmed by microsatellite genotyping. (F) Representative telomere FISH images. Blue, chromosomes stained with DAPI; green dots, telomeres. (G) Histogram displaying distribution of relative telomere length shown as TFU by telomere Q-FISH. Medium telomere length (green lines) is shown as mean ± SD above each panel. See also Figure S7.