Brief report: impaired cell reprogramming in nonhomologous end joining deficient cells

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Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems
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Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems

Published on: February 2, 2024

F Javier Molina-Estevez1, M Luz Lozano, Susana Navarro

  • 1Division of Hematopoietic Innovative Therapies (HIT), Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT)/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBER-ER), Madrid, Spain.

Stem Cells (Dayton, Ohio)
|May 1, 2013
PubMed

Insights

DNA repair proteins are crucial for cell reprogramming. DNA-PKcs deficiency impairs induced pluripotent stem cell generation, highlighting DNA damage response roles in reprogramming efficiency.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genetics

Background:

  • Growing interest in DNA damage response (DDR) mechanisms during cell reprogramming.
  • The role of nonhomologous end joining (NHEJ) proteins in reprogramming is largely unexplored.
  • NHEJ is a primary pathway for repairing DNA double-strand breaks.

Purpose of the Study:

  • To investigate the role of DNA-PKcs, a key NHEJ protein, in the reprogramming of mouse embryonic fibroblasts (MEFs).
  • To assess the impact of DNA-PKcs deficiency on induced pluripotent stem cell (iPSC) generation and stability.

Main Methods:

  • Reprogramming of primary MEFs from severe combined immunodeficient (Scid) mice (defective in DNA-PKcs) and wild-type (wt) mice using lentiviral vectors (LV).
  • Analysis of reprogramming efficiency, iPSC colony formation, senescence markers (SA-β-Gal, P16/INK(4a)), proliferation rates, and transduction efficacy.
  • Utilized Sleeping Beauty transposon/transposase systems to isolate DNA-PKcs-deficient iPSCs.

Main Results:

  • Scid MEFs exhibited significantly lower reprogramming efficiency (4-7 fold reduction) compared to wt MEFs.
  • Scid iPSC-like clones showed premature loss and spontaneous differentiation.
  • Increased senescence markers (SA-β-Gal, P16/INK(4a)) were observed in Scid MEFs during reprogramming, without affecting proliferation or transduction rates.
  • DNA-PKcs-deficient iPSCs were successfully isolated using improved transposon systems, retaining parental genotype and radiation hypersensitivity.

Conclusions:

  • DNA-PKcs deficiency impairs reprogramming efficiency and iPSC stability, likely due to increased senescence.
  • This study establishes a novel disease-specific iPSC model for studying DNA-PKcs mutations.
  • Findings offer insights into DDR's role in reprogramming and potential improvements for cell/gene therapies.
Keywords:
DNA-dependent protein kinase catalytic subunitImmunodeficient mouseInduced pluripotent stem cellsNonhomologous end joiningReprogrammingSevere combined immunodeficiency

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