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Telomere length is regulated by FGF-2 in human embryonic stem cells and affects the life span of its differentiated progenies

Abstract

The ability of human embryonic stem cells (hESCs) to proliferate indefinitely is attributed to its high telomerase activity and associated long telomere. However, factors regulating telomere length in hESCs remain largely uncharacterized. The aims of this study were, to identify factors which modulate telomere length of hESCs, and to determine if the telomere length of hESCs influences cellular senescence of its differentiated progeny cells. Telomerase reverse transcriptase (TERT) gene expression, telomerase activity and telomere length of hESCs cultured in different culture systems were compared. Genetically identical hESCs of different telomere lengths were differentiated into fibroblasts simultaneously, and the population doubling and cellular senescence levels were determined. We found that telomere lengths were significantly different in different culture systems and Fibroblast growth factor-2 (FGF-2) upregulated TERT expression, telomerase activity and telomere length via Wnt/β-catenin signaling pathway in hESCs in a significant manner. We also provide evidence that fibroblast differentiated from hESCs with longer telomere exhibited significant more population doublings and longer life span than those derived from hESCs with shorter telomeres. Thus, FGF-2 levels in hESCs culture systems can be manipulated to generate cells with longer telomere which would be advantageous in the applications of hESCs in regenerative medicine.

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Acknowledgments

This research was supported by grant from National University Health System (R221000085733) and National University of Singapore (R221000050133).

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Correspondence to Tong Cao.

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a

Fig. S1 Southern blot image of TRF assay of H1 hESCs cultured in MEF systems at different passages. The band on top of the second and third lane is contaminated telomere of MEF feeder. b Southern blot image of TRF assay of H1 hESCs cultured in mTeSR system at different passages. Yellow lines indicate average telomere length of respective samples. c Southern blot image of TRF assay of MEF feeder cells only (Lane 5) and hESCs cultured in mTeSR system (Lane 6) and hESCs cultured in MEF feeder system (Lane 2, 3 and 4)

a

Fig. S2 Relative gene expression of TERT after H1 hESCs were treated 24 h with and without TGFβ (0.6 ng/mL) in CM system. b Relative gene expression of TERT after H1 hESCs were treated 24 h with or without LiCl (1 and 5 mM) in CM system. Data were presented as mean ± SEM and results were generated from three independent experiments, n.s. not significant. Compared with non-treated H1 hESCs. c Western blot repeats of active β-catenin and total β-catenin in H1 hESCs were treated with either FGF-2 (100 ng/mL), DKK1 (50 ng/mL) + FGF-2 (100 ng/mL), DKK1 (50 ng/mL), DKK1 (50 ng/mL) + Wnt3a (100 ng/mL) or Wnt3a (100 ng/mL) for 24 h in CM system. d Western blot of active β-catenin and total β-catenin in H1 hESCs treated with different level of FGF-2. e Relative quantification of above western blot. f Relative TA of H1 hESCs treated with FGF-2 of different concentration. Data were presented as mean ± SEM and results were generated from three independent experiments, n.s. not significant. Compared with non-treated H1 hESCs

a

Fig. S3 Relative gene expression of TERT of long-ebFs and short-ebFs at the indicated passage numbers. b Relative gene expression of the fibroblast marker P4Hβ of ebFs with long- and short telomeres at the indicated passage numbers. Data were presented as mean ± SEM and results were generated from three independent experiments, Asterisk p < 0.05, compared with undifferentiated H1 hESCs. n.s. not significant. c Relative gene expression of p21 of ebFs with long- and short-telomere at the indicated passage number. Data were presented as mean ± SEM and results were generated from three independent experiments, Asterisk p < 0.05, compared with undifferentiated H1 hESCs. n.s. not significant

Table S1 Primer sequences used for RT-PCR and real-time qPCR in this study

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Zou, Y., Tong, H.J., Li, M. et al. Telomere length is regulated by FGF-2 in human embryonic stem cells and affects the life span of its differentiated progenies. Biogerontology 18, 69–84 (2017). https://doi.org/10.1007/s10522-016-9662-8

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Keywords

  • hESC
  • Telomere
  • Telomerase
  • Cellular senescence
  • Wnt