Differentiation of human induced pluripotent or embryonic stem cells decreases the DNA damage response

Abstract Genomic integrity is critical for preservation of stem cell function and is, maintained through a robust DNA damage response (DDR) with systemized DNA DSB repair by either the non-homologous end joining (NHEJ) pathway or the homologous recombination (HR) pathway. To examine DDR during stem cell differentiation, human embryonic (hES) and induced pluripotent stem (IPS) cells were exposed to DNA damaging agents and DNA damage signaling/repair measured. Differentiated cells displayed a higher frequency of residual DNA damage, chromosomal aberrations, cells with delayed γ-H2AX foci disappearance and a reduced number of RAD51 foci. Factors impacting DNA DSB repair by HR formed reduced foci in differentiated cells. The reduction in repairosome foci formation after DNA damage was not due to changes in HR protein levels, which were unchanged by differentiation. Differentiated cells also displayed a higher frequency of stalled DNA replication forks and decreased firing of new replication origins from transient inhibition of DNA synthesis by hydroxyurea treatment. In addition, we observed that differentiated cells exhibit a higher frequency of R-loops. A similar decline in DDR was observed as early stage mouse astrocytes differentiated into later stage astrocytes. Our studies thus suggest that DSB repair by homologous recombination is increasingly impaired during stem cell differentiation while the NHEJ pathway is minimally altered.