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Cell Death & Differentiation
  • Original Paper
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A human iPSC model of Ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells and emerging haematopoietic progenitors

Cell Death & Differentiationvolume 20, pages1089–1100 (2013)Cite this article

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Abstract

DNA double strand breaks (DSBs) are the most common form of DNA damage and are repaired by non-homologous-end-joining (NHEJ) or homologous recombination (HR). Several protein components function in NHEJ, and of these, DNA Ligase IV is essential for performing the final ‘end-joining’ step. Mutations inDNA Ligase IV result in LIG4 syndrome, which is characterised by growth defects, microcephaly, reduced number of blood cells, increased predisposition to leukaemia and variable degrees of immunodeficiency. In this manuscript, we report the creation of a human induced pluripotent stem cell (iPSC) model of LIG4 deficiency, which accurately replicates the DSB repair phenotype of LIG4 patients. Our findings demonstrate that impairment of NHEJ-mediated-DSB repair in human iPSC results in accumulation of DSBs and enhanced apoptosis, thus providing new insights into likely mechanisms used by pluripotent stem cells to maintain their genomic integrity. Defects in NHEJ-mediated-DSB repair also led to a significant decrease in reprogramming efficiency of human cells and accumulation of chromosomal abnormalities, suggesting a key role for NHEJ in somatic cell reprogramming and providing insights for future cell based therapies for applications of LIG4-iPSCs. Although haematopoietic specification of LIG4-iPSC is not affectedper se, the emerging haematopoietic progenitors show a high accumulation of DSBs and enhanced apoptosis, resulting in reduced numbers of mature haematopoietic cells. Together our findings provide new insights into the role of NHEJ-mediated-DSB repair in the survival and differentiation of progenitor cells, which likely underlies the developmental abnormalities observed in many DNA damage disorders. In addition, our findings are important for understanding how genomic instability arises in pluripotent stem cells and for defining appropriate culture conditions that restrict DNA damage and result inex vivo expansion of stem cells with intact genomes.

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Abbreviations

NHEJ:

Non homologous end joining

DSB:

double strand break

iPSC:

induced pluripotent stem cells

HR:

homologous recombination

IR:

ionising radiation

MMEJ:

microhomology-mediated-end joining

ESC:

embryonic stem cells

References

  1. Hartlerode AJ, Scully R . Mechanisms of double-strand break repair in somatic mammalian cells.Biochem J 2009;423: 157–168.

    Article CAS  Google Scholar 

  2. Jeggo PA . DNA breakage and repair.Adv Genet 1998;38: 185–218.

    Article CAS  Google Scholar 

  3. Holthausen JT, Wyman C, Kanaar R . Regulation of DNA strand exchange in homologous recombination.DNA Repair (Amst) 2010;9: 1264–1272.

    Article CAS  Google Scholar 

  4. Lieber MR, Ma Y, Pannicke U, Schwarz K . Mechanism and regulation of human non-homologous DNA end-joining.Nat Rev Mol Cell Biol 2003;4: 712–720.

    Article CAS  Google Scholar 

  5. Grawunder U, Grawunder U, Wilm M, Wu M, Kulesza P, Wilson TEet al. Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells.Nature 1997;388: 492–495.

    Article CAS  Google Scholar 

  6. Ahnesorg P, Smith P, Jackson SP . XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining.Cell 2006;124: 301–313.

    Article CAS  Google Scholar 

  7. Nussenzweig A, Nussenzweig MC . A backup DNA repair pathway moves to the forefront.Cell 2007;131: 223–225.

    Article CAS  Google Scholar 

  8. Jackson SP, Bartek J . The DNA-damage response in human biology and disease.Nature 2009;461: 1071–1078.

    Article CAS  Google Scholar 

  9. O'Driscoll M, Gennery AR, Seidel J, Concannon P, Jeggo PA . An overview of three new disorders associated with genetic instability: LIG4 syndrome, RS-SCID and ATR-Seckel syndrome.DNA Repair (Amst) 2004;3: 1227–1235.

    Article CAS  Google Scholar 

  10. Frank KM, Sekiguchi JM, Seidl KJ, Swat W, Rathbun GA, Cheng HLet al. Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV.Nature 1998;396: 173–177.

    Article CAS  Google Scholar 

  11. Frank KM, Sharpless NE, Gao Y, Sekiguchi JM, Ferguson DO, Zhu Cet al. DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway.Mol Cell 2000;5: 993–1002.

    Article CAS  Google Scholar 

  12. Gao Y, Sun Y, Frank KM, Dikkes P, Fujiwara Y, Seidl KJet al. A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis.Cell 1998;95: 891–902.

    Article CAS  Google Scholar 

  13. Nijnik A, Woodbine L, Marchetti C, Dawson S, Lambe T, Liu Cet al. DNA repair is limiting for haematopoietic stem cells during ageing.Nature 2007;447: 686–690.

    Article CAS  Google Scholar 

  14. Gatz SA, Ju L, Gruber R, Hoffmann E, Carr AM, Wang ZQet al. Requirement for DNA ligase IV during embryonic neural development.J Neurosci 2011;31: 10088–10100.

    Article CAS  Google Scholar 

  15. O'Driscoll M, Cerosaletti KM, Girard PM, Dai Y, Stumm M, Kysela Bet al. DNA ligase IV mutations identified in patients exhibiting developmental delay and immunodeficiency.Mol Cell 2001;8: 1175–1185.

    Article CAS  Google Scholar 

  16. Riballo E, Critchlow SE, Teo SH, Doherty AJ, Priestley A, Broughton Bet al. Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient.Curr Biol 1999;9: 699–702.

    Article CAS  Google Scholar 

  17. Ben-Omran TI, Cerosaletti K, Concannon P, Weitzman S, Nezarati MM . A patient with mutations in DNA Ligase IV: clinical features and overlap with Nijmegen breakage syndrome.Am J Med Genet A 2005;137A: 283–287.

    Article  Google Scholar 

  18. Chistiakov DA Ligase IV syndrome chapter 16: 175-183: Diseases of DNA repair, edited by S. I. Ahmad.

  19. Rossi DJ, Bryder D, Seita J, Nussenzweig A, Hoeijmakers J, Weissman IL . Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age.Nature 2007;447: 725–729.

    Article CAS  Google Scholar 

  20. Zhang S, Yajima H, Huynh H, Zheng J, Callen E, Chen HTet al. Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair.J Cell Biol 2011;193: 295–305.

    Article CAS  Google Scholar 

  21. Lucas D, Escudero B, Ligos JM, Segovia JC, Estrada JC, Terrados Get al. Altered hematopoiesis in mice lacking DNA polymerase mu is due to inefficient double-strand break repair.PLoS Genet 2009;5: e1000389.

    Article  Google Scholar 

  22. Nijnik A, Dawson S, Crockford TL, Woodbine L, Visetnoi S, Bennett Set al. Impaired lymphocyte development and antibody class switching and increased malignancy in a murine model of DNA ligase IV syndrome.J Clin Invest 2009;119: 1696–1705.

    Article CAS  Google Scholar 

  23. Rucci F, Notarangelo LD, Fazeli A, Patrizi L, Hickernell T, Paganini Tet al. Homozygous DNA ligase IV R278H mutation in mice leads to leaky SCID and represents a model for human LIG4 syndrome.Proc Natl Acad Sci USA 2010;107: 3024–3029.

    Article CAS  Google Scholar 

  24. Mohrin M, Bourke E, Alexander D, Warr MR, Barry-Holson K, Le Beau MMet al. Hematopoietic stem cell quiescence promotes error-prone DNA repair and mutagenesis.Cell Stem Cell 2010;7: 174–185.

    Article CAS  Google Scholar 

  25. Saretzki G, Armstrong L, Leake A, Lako M, von Zglinicki T . Stress defense in murine embryonic stem cells is superior to that of various differentiated murine cells.Stem Cells 2004;22: 962–971.

    Article CAS  Google Scholar 

  26. Saretzki G, Walter T, Atkinson S, Passos JF, Bareth B, Keith WNet al. Downregulation of multiple stress defence mechanisms during differentiation of human embryonic stem cells.Stem Cells 2008;26: 455–464.

    Article CAS  Google Scholar 

  27. Bogomazova AN, Lagarkova MA, Tskhovrebova LV, Shutova MV, Kiselev SL . Error-prone nonhomologous end joining repair operates in human pluripotent stem cells during late G2.Aging 2011;3: 584–596.

    Article  Google Scholar 

  28. Fan J, Robert C, Jang YY, Liu H, Sharkis S, Baylin SBet al. Human induced pluripotent cells resemble embryonic stem cells demonstrating enhanced levels of DNA repair and efficacy of nonhomologous end-joining.Mutat Res 2011;713: 8–17.

    Article CAS  Google Scholar 

  29. Roddam PL, Rollinson S, O'Driscoll M, Jeggo PA, Jack A, Morgan GJ . Genetic variants of NHEJ DNA ligase IV can affect the risk of developing multiple myeloma, a tumour characterised by aberrant class switch recombination.J Med Genet 2002;39: 900–905.

    Article CAS  Google Scholar 

  30. Marión RM, Strati K, Li H, Murga M, Blanco R, Ortega Set al. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity.Nature 2009;460: 1149–1153.

    Article  Google Scholar 

  31. Seluanov A, Mao Z, Gorbunova V . Analysis of DNA double-strand break (DSB) repair in mammalian cells.J Vis Exp 2002;43 pii 2002.

    Google Scholar 

  32. Delacôte F, Han M, Stamato TD, Jasin M, Lopez BS . An xrcc4 defect or Wortmannin stimulates homologous recombination specifically induced by double-strand breaks in mammalian cells.Nuc Acids Res 2002;30: 3454–3463.

    Article  Google Scholar 

  33. Pierce AJ, Hu P, Han M, Ellis N, Jasin M . Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells.Genes Dev 2001;15: 3237–3242.

    Article CAS  Google Scholar 

  34. Ohgushi M, Matsumura M, Eiraku M, Murakami K, Aramaki T, Nishiyama Aet al. Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells.Cell Stem Cell 2010;7: 225–239.

    Article CAS  Google Scholar 

  35. Chen G, Hou Z, Gulbranson DR, Thomson JA . Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells.Cell Stem Cell 2010;7: 240–248.

    Article CAS  Google Scholar 

  36. Kennedy M, D'Souza SL, Lynch-Kattman M, Schwantz S, Keller G . Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures.Blood 2007;109: 2679–2687.

    CAS PubMed PubMed Central  Google Scholar 

  37. Yung S, Ledran M, Moreno-Gimeno I, Conesa A, Montaner D, Dopazo Jet al. Large-scale transcriptional profiling and functional assays reveal important roles for Rho-GTPase signalling and SCL during haematopoietic differentiation of human embryonic stem cells.Hum Mol Genet 2011;20: 4932–4946.

    Article CAS  Google Scholar 

  38. Real PJ, Ligero G, Ayllon V, Ramos-Mejia V, Bueno C, Gutierrez-Aranda Iet al. SCL/TAL1 regulates hematopoietic specification from human embryonic stem cells.Mol Ther 2012;20: 1443–1453.

    Article CAS  Google Scholar 

  39. Bañuelos CA, Banáth JP, MacPhail SH, Zhao J, Eaves CA, O'Connor MDet al. Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks.DNA Repair (Amst) 2008;7: 1471–1483.

    Article  Google Scholar 

  40. Armstrong L, Tilgner K, Saretzki G, Atkinson SP, Stojkovic M, Moreno Ret al. Human induced pluripotent stem cell lines show stress defense mechanisms and mitochondrial regulation similar to those of human embryonic stem cells.Stem Cells 2010;28: 661–673.

    Article CAS  Google Scholar 

  41. Mayshar Y, Ben-David U, Lavon N, Biancotti JC, Yakir B, Clark ATet al. Identification and classification of chromosomal aberrations in human induced pluripotent stem cells.Cell Stem Cell 2010;7: 521–531.

    Article CAS  Google Scholar 

  42. Elliott AM, Elliott KA, Kammesheidt A . High resolution array-CGH characterization of human stem cells using a stem cell focused microarray.Mol Biotech 2010;46: 234–242.

    Article CAS  Google Scholar 

  43. Laurent LC, Ulitsky I, Slavin I, Tran H, Schork A, Morey Ret al. Dynamic changes in the copy number of pluripotency and cell proliferation genes in human ESCs and iPSCs during reprogramming and time in culture.Cell Stem Cell 2011;8: 106–118.

    Article CAS  Google Scholar 

  44. International Stem Cell Initiative Amps K, Andrews PW, Anyfantis G, Armstrong L, Avery S, Baharvand Het al. Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage.Nat Biotech 2011;29: 1132–1144.

    Article  Google Scholar 

  45. Martins-Taylor K, Nisler BS, Taapken SM, Compton T, Crandall L, Montgomery KDet al. Recurrent copy number variations in human induced pluripotent stem cells.Nat Biotech 2011;29: 488–491.

    Article CAS  Google Scholar 

  46. Yung SK, Tilgner K, Ledran MH, Habibollah S, Neganova I, Singhapol Cet al. Human pluripotent stem cell models of Fanconi Anaemia deficiency reveal an important role for Fanconi Anaemia proteins in cellular reprogramming and survival of haematopoietic progenitors.Stem Cells 2013;31: 1022–1029.

    Article CAS  Google Scholar 

  47. Müller LU, Milsom MD, Harris CE, Vyas R, Brumme KM, Parmar Ket al. Overcoming reprogramming resistance of Fanconi anemia cells.Blood 2012;119: 5449–5457.

    Article  Google Scholar 

  48. Stojkovic M, Lako M, Stojkovic P, Stewart R, Przyborski S, Armstrong Let al. Derivation of human embryonic stem cells from day-8 blastocysts recovered after three-stepin vitro culture.Stem Cells 2004;22: 790–797.

    Article  Google Scholar 

  49. Neganova I, Zhang X, Atkinson S, Lako M . Expression and functional analysis of G1 to S regulatory components reveals an important role for CDK2 in cell cycle regulation in human embryonic stem cells.Oncogene 2008;28: 20–30.

    Article  Google Scholar 

  50. Mellough CB, Sernagor E, Moreno-Gimeno I, Steel DH, Lako M . Efficient stage-specific differentiation of human pluripotent stem cells toward retinal photoreceptor cells.Stem Cells 2012;30: 673–686.

    Article CAS  Google Scholar 

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Acknowledgements

The authors would like to thank Mr Ian Dimmick and Dr Owen Hughes for their help with the flow cytometric analysis, Dr. Josef Jaros for help with the work on cytoskeleton and ROCK signalling, Complement Genomics plc. for carrying out the DNA fingerprinting analysis, Addgene for provision of the Cre construct and Coriell Cell Repositories for providing some of the LIG4 patient fibroblasts. This study was supported by Leukamia and Lymphoma Research Grant 09005 and funds for research in the field of Regenerative Medicine through the collaboration agreement from the Conselleria de Sanidad (Generalitat Valenciana) and the Instituto de Salud Carlos III (Ministry of Science and Innovation), Spain.

Author information

Authors and Affiliations

  1. Institute of Genetic Medicine, International Centre for Life, Newcastle University, Newcastle, UK

    K Tilgner, I Neganova, S Yung, J Evans, L Armstrong & M Lako

  2. NESCI, Newcastle University, Newcastle, UK

    K Tilgner, I Neganova, S Yung, L Armstrong & M Lako

  3. Centro de Investigacion Principe Felipe, Valencia, Spain

    I Moreno-Gimeno & D Burks

  4. Princess Al Jawhara Al-Brahim Center of Excellence in Research of Hereditary Disorders, King Abdulaziz University, Jeddah, Saudi Arabia

    J Y AL-Aama

  5. Institute for Ageing and Health, Newcastle University, Newcastle, UK

    C Singhapol & G Saretzki

  6. Department of Biology, University of Rochester, Rochester, NY, USA

    V Gorbunova

  7. Institute of Cellular Medicine, Newcastle University, Newcastle, UK

    A Gennery

  8. School of Biological and Biomedical Sciences, Durham University, Durham, UK

    S Przyborski

  9. Human Genetics Department, Medical Faculty, University of Kragujevac, Kragujevac, Serbia

    M Stojkovic

  10. Genome Damage and Stability Centre, University of Sussex, Brighton, UK

    P Jeggo

Authors
  1. K Tilgner
  2. I Neganova
  3. I Moreno-Gimeno
  4. J Y AL-Aama
  5. D Burks
  6. S Yung
  7. C Singhapol
  8. G Saretzki
  9. J Evans
  10. V Gorbunova
  11. A Gennery
  12. S Przyborski
  13. M Stojkovic
  14. L Armstrong
  15. P Jeggo
  16. M Lako

Corresponding author

Correspondence toM Lako.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Edited by B Zhivotovsky

Author contributions:

Katarzyna Tilgner: performed the majority of experiments, data collection and analysis, contribution to manuscript writing, final approval of manuscript. Irina Neganova, Inmaculada Moreno-Gimeno, Deborah Burks, Sun Yung, Jerome Evans, Gabriele Saretzki, Chatchawan Singhapol: performed some of the experiments, final approval of manuscript.Jumana Yousuf AL-Aama: provided important reagents for this work, performed some of the experiments, final approval of manuscript. Vera Gorbunova: provided important reagents for this work, final approval of manuscript. Andrew Gennery: conception and design of the study, provided important reagents for this work, final approval of manuscript. Stefan Przyborski: performed some of the experiments, collection and analysis of the data, contributed to manuscript writing and final approval of manuscript. Miodrag Stojkovic, Lyle Armstrong: conception and design, manuscript writing, fund raising and final approval of manuscript. Penny Jeggo: provided reagents for this work, design, data analysis, manuscript writing, fund raising and final approval of manuscript. Majlinda Lako: conception and design, performed experiments, data analysis, manuscript writing, fund raising and final approval of manuscript.

Supplementary Information accompanies this paper on Cell Death and Differentiation website

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Tilgner, K., Neganova, I., Moreno-Gimeno, I.et al. A human iPSC model of Ligase IV deficiency reveals an important role for NHEJ-mediated-DSB repair in the survival and genomic stability of induced pluripotent stem cells and emerging haematopoietic progenitors.Cell Death Differ20, 1089–1100 (2013). https://doi.org/10.1038/cdd.2013.44

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