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Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells

Abstract

The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human development and disease1,2,3,4,5,6. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis7,8. Short telomeres, a hallmark of dyskeratosis congenita, impair tissue stem cell function in mouse models, indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita9,10. Here we show that even in the undifferentiated state, iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT, the telomerase reverse transcriptase, a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast, mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53), telomerase catalytic activity is unperturbed, yet the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal, indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics.

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Figure 1: Dyskeratosis congenita iPSCs with heterozygous TERT mutations show reduced telomerase levels.
Figure 2: Preserved activity, but pronounced mislocalization of telomerase in TCAB1 -mutant iPSCs.
Figure 3: Diminished TERC levels, reduced activity and impaired assembly of mature telomerase in X-linked dyskeratosis congenita iPSCs.
Figure 4: Impaired telomere maintenance and loss of self-renewal in dyskeratosis congenita iPSCs.

References

  1. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007)

    CAS  Article  Google Scholar 

  2. Rashid, S. T. et al. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J. Clin. Invest. 120, 3127–3136 (2010)

    CAS  Article  Google Scholar 

  3. Liu, G. H. et al. Recapitulation of premature ageing with iPSCs from Hutchinson–Gilford progeria syndrome. Nature advance online publication. 10.1038/nature09879 (23 February 2011)

  4. Soldner, F. et al. Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136, 964–977 (2009)

    CAS  Article  Google Scholar 

  5. Dimos, J. T. et al. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321, 1218–1221 (2008)

    ADS  CAS  Article  Google Scholar 

  6. Maehr, R. et al. Generation of pluripotent stem cells from patients with type 1 diabetes. Proc. Natl Acad. Sci. USA 106, 15768–15773 (2009)

    ADS  CAS  Article  Google Scholar 

  7. Walne, A. J. & Dokal, I. Advances in the understanding of dyskeratosis congenita. Br. J. Haematol. 145, 164–172 (2009)

    CAS  Article  Google Scholar 

  8. Alter, B. P. et al. Malignancies and survival patterns in the National Cancer Institute inherited bone marrow failure syndromes cohort study. Br. J. Haematol. 150, 179–188 (2010)

    PubMed  PubMed Central  Google Scholar 

  9. Lee, H. W. et al. Essential role of mouse telomerase in highly proliferative organs. Nature 392, 569–574 (1998)

    ADS  CAS  Article  Google Scholar 

  10. Allsopp, R. C., Morin, G. B., DePinho, R., Harley, C. B. & Weissman, I. L. Telomerase is required to slow telomere shortening and extend replicative lifespan of HSCs during serial transplantation. Blood 102, 517–520 (2003)

    CAS  Article  Google Scholar 

  11. Bessler, M., Wilson, D. B. & Mason, P. J. Dyskeratosis congenita. FEBS Lett. 584, 3831–3838 (2010)

    CAS  Article  Google Scholar 

  12. Mitchell, J. R., Wood, E. & Collins, K. A telomerase component is defective in the human disease dyskeratosis congenita. Nature 402, 551–555 (1999)

    ADS  CAS  Article  Google Scholar 

  13. Zhong, F. et al. Disruption of telomerase trafficking by TCAB1 mutation causes dyskeratosis congenita. Genes Dev. 25, 11–16 (2011)

    CAS  Article  Google Scholar 

  14. Alter, B. P. et al. Very short telomere length by flow fluorescence in situ hybridization identifies patients with dyskeratosis congenita. Blood 110, 1439–1447 (2007)

    CAS  Article  Google Scholar 

  15. Agarwal, S. et al. Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients. Nature 464, 292–296 (2010)

    ADS  CAS  Article  Google Scholar 

  16. Marion, R. M. et al. Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells. Cell Stem Cell 4, 141–154 (2009)

    CAS  Article  Google Scholar 

  17. Stadtfeld, M., Maherali, N., Breault, D. T. & Hochedlinger, K. Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2, 230–240 (2008)

    CAS  Article  Google Scholar 

  18. Yoshida, Y., Takahashi, K., Okita, K., Ichisaka, T. & Yamanaka, S. Hypoxia enhances the generation of induced pluripotent stem cells. Cell Stem Cell 5, 237–241 (2009)

    CAS  Article  Google Scholar 

  19. Wong, J. M. & Collins, K. Telomerase RNA level limits telomere maintenance in X-linked dyskeratosis congenita. Genes Dev. 20, 2848–2858 (2006)

    CAS  Article  Google Scholar 

  20. Zaug, A. J., Podell, E. R., Nandakumar, J. & Cech, T. R. Functional interaction between telomere protein TPP1 and telomerase. Genes Dev. 24, 613–622 (2010)

    CAS  Article  Google Scholar 

  21. Vulliamy, T. et al. Disease anticipation is associated with progressive telomere shortening in families with dyskeratosis congenita due to mutations in TERC. Nature Genet. 36, 447–449 (2004)

    CAS  Article  Google Scholar 

  22. Armanios, M. et al. Haploinsufficiency of telomerase reverse transcriptase leads to anticipation in autosomal dominant dyskeratosis congenita. Proc. Natl Acad. Sci. USA 102, 15960–15964 (2005)

    ADS  CAS  Article  Google Scholar 

  23. Venteicher, A. S. et al. A human telomerase holoenzyme protein required for Cajal body localization and telomere synthesis. Science 323, 644–648 (2009)

    ADS  CAS  Article  Google Scholar 

  24. Tycowski, K. T., Shu, M. D., Kukoyi, A. & Steitz, J. A. A conserved WD40 protein binds the Cajal body localization signal of scaRNP particles. Mol. Cell 34, 47–57 (2009)

    CAS  Article  Google Scholar 

  25. Lundblad, V. & Szostak, J. W. A mutant with a defect in telomere elongation leads to senescence in yeast. Cell 57, 633–643 (1989)

    CAS  Article  Google Scholar 

  26. Matera, A. G., Terns, R. M. & Terns, M. P. Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs. Nature Rev. Mol. Cell Biol. 8, 209–220 (2007)

    CAS  Article  Google Scholar 

  27. Byrne, J. A., Nguyen, H. N. & Reijo Pera, R. A. Enhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblasts. PLoS ONE 4, e7118 (2009)

    ADS  Article  Google Scholar 

  28. Sommer, C. A. et al. Induced pluripotent stem cell generation using a single lentiviral stem cell cassette. Stem Cells 27, 543–549 (2009)

    CAS  Article  Google Scholar 

  29. Tomlinson, R. L. et al. Telomerase reverse transcriptase is required for the localization of telomerase RNA to Cajal bodies and telomeres in human cancer cells. Mol. Biol. Cell 19, 3793–3800 (2008)

    CAS  Article  Google Scholar 

  30. Middleman, E. J., Choi, J., Venteicher, A. S., Cheung, P. & Artandi, S. E. Regulation of cellular immortalization and steady-state levels of the telomerase reverse transcriptase through its carboxy-terminal domain. Mol. Cell. Biol. 26, 2146–2159 (2006)

    CAS  Article  Google Scholar 

  31. Cristofari, G. & Lingner, J. Telomere length homeostasis requires that telomerase levels are limiting. EMBO J. 25, 565–574 (2006)

    CAS  Article  Google Scholar 

  32. Lei, M., Zaug, A. J., Podell, E. R. & Cech, T. R. Switching human telomerase on and off with hPOT1 protein in vitro. J. Biol. Chem. 280, 20449–20456 (2005)

    CAS  Article  Google Scholar 

  33. Wang, F. et al. The POT1–TPP1 telomere complex is a telomerase processivity factor. Nature 445, 506–510 (2007)

    ADS  CAS  Article  Google Scholar 

  34. Venteicher, A. S., Meng, Z., Mason, P. J., Veenstra, T. D. & Artandi, S. E. Identification of ATPases pontin and reptin as telomerase components essential for holoenzyme assembly. Cell 132, 945–957 (2008)

    CAS  Article  Google Scholar 

  35. Mochizuki, Y., He, J., Kulkarni, S., Bessler, M. & Mason, P. J. Mouse dyskerin mutations affect accumulation of telomerase RNA and small nucleolar RNA, telomerase activity, and ribosomal RNA processing. Proc. Natl Acad. Sci. USA 101, 10756–10761 (2004)

    ADS  CAS  Article  Google Scholar 

  36. Seabright, M. Rapid banding technique for human chromosomes. Lancet 2, 971–972 (1971)

    CAS  Article  Google Scholar 

  37. Deb-Rinker, P., Ly, D., Jezierski, A., Sikorska, M. & Walker, P. R. Sequential DNA methylation of the Nanog and Oct-4 upstream regions in human NT2 cells during neuronal differentiation. J. Biol. Chem. 280, 6257–6260 (2005)

    CAS  Article  Google Scholar 

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Acknowledgements

L.F.Z.B. is the recipient of a Pew Fellowship. M.F.P. and K.T.X. are the recipients of NSF Graduate Research Fellowships. F.L.Z. was supported by A*STAR, Singapore. We thank the patients for their valuable contributions and L. Leathwood for study support. This work was supported, in part, by the intramural research program of the Division of Cancer Epidemiology and Genetics, NCI, NIH; by a CIRM Shared Research Laboratory grant to R.R.P.; and by grants from the NCI, NIA, NHLBI and CIRM to S.E.A.

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L.F.Z.B., M.F.P., F.L.Z and S.E.A. designed the experiments and analysed the data; L.F.Z.B., M.F.P., F.L.Z., H.N.N., K.T.X., A.J.Z., S.M.C., J.C., V.S. and A.C. performed the experiments; N.G., M.W., B.P.A., T.R.C., S.A.S. and R.A.R.P. analysed the data; N.G., B.P.A and S.A.S. collected patients’ material; L.F.Z.B. and S.E.A. wrote the manuscript.

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Correspondence to Steven E. Artandi.

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Batista, L., Pech, M., Zhong, F. et al. Telomere shortening and loss of self-renewal in dyskeratosis congenita induced pluripotent stem cells. Nature 474, 399–402 (2011). https://doi.org/10.1038/nature10084

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