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Differential role of nonhomologous end joining factors in the generation, DNA damage response, and myeloid differentiation of human induced pluripotent stem cells

Significance In this study, we compare the differential impact of nonhomologous end-joining–deficient induced pluripotent stem cells (iPSCs) derived from patients with mutations in genes encoding DNA ligase 4, DNA-protein kinase catalytic subunit, or Artemis, on cellular reprogramming, DNA damage repair, and myeloid differentiation. We found a predominant role for ligase 4 in genomic stability and myeloid differentiation of iPSCs, but a minor one for Artemis. Disease modeling with patient-derived iPSCs may provide significant insights into phenotypic variability associated with mutations in various genes along the same pathway and may serve as a platform to explore tissue-specific consequences of single gene defects and development of therapeutic interventions. Nonhomologous end-joining (NHEJ) is a key pathway for efficient repair of DNA double-strand breaks (DSBs) and V(D)J recombination. NHEJ defects in humans cause immunodeficiency and increased cellular sensitivity to ionizing irradiation (IR) and are variably associated with growth retardation, microcephaly, and neurodevelopmental delay. Repair of DNA DSBs is important for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). To compare the specific contribution of DNA ligase 4 (LIG4), Artemis, and DNA-protein kinase catalytic subunit (PKcs) in this process and to gain insights into phenotypic variability associated with these disorders, we reprogrammed patient-derived fibroblast cell lines with NHEJ defects. Deficiencies of LIG4 and of DNA-PK catalytic activity, but not Artemis deficiency, were associated with markedly reduced reprogramming efficiency, which could be partially rescued by genetic complementation. Moreover, we identified increased genomic instability in LIG4-deficient iPSCs. Cell cycle synchronization revealed a severe defect of DNA repair and a G0/G1 cell cycle arrest, particularly in LIG4- and DNA-PK catalytically deficient iPSCs. Impaired myeloid differentiation was observed in LIG4-, but not Artemis- or DNA-PK–mutated iPSCs. These results indicate a critical importance of the NHEJ pathway for somatic cell reprogramming, with a major role for LIG4 and DNA-PKcs and a minor, if any, for Artemis.