When we compared the DDR in stem cells before and after differentiation, we found that differentiated stem cells have the following: (1) higher frequency of spontaneous chromosome aberrations; (2) reduced DNA DSB repair after IR exposure; (3) higher frequency of S-phase-specific IR-induced chromosome aberrations; (4) higher frequency of residual -H2AX foci formation after IR exposure or cisplatin treatment; (5) higher frequency of cells with 53BP1 and RIF1 co-localization; and (6) higher frequency of cells with a reduced number of RAD51 or BRCA1 foci after IR exposure or cisplatin treatment compared with undifferentiated stem cells. cell that is terminally differentiated. Various factors, including reactive oxygen species, that accumulate during differentiation and over the stem cell lifespan, can cause DNA damage (Mikhed et?al., 2015). In addition, differentiation-dependent changes in chromatin structure and transcriptional alterations (Nashun et?al., 2015, Tran et?al., 2015) can also affect genomic integrity by altering the DNA damage response (DDR) and repair facility. Thus, genomic stability is likely to be under increased stress during differentiation. How factors that induce differentiation, such as NO donors, affect stem cell genomic stability is usually unclear. Stem cells benefit throughout their lifetime from a strong DNA damage repair activity that enhances resilience toward various MCC950 sodium environmental factors. Indeed, somatic cells and stem cells differ significantly in their radio-sensitivity (Chlon et?al., 2016, Maynard et?al., 2008, Lan et?al., 2012, Momcilovic et?al., 2009, Wilson et?al., 2010). However, it is not known how DNA double-strand break (DSB) repair mechanisms are affected during stem cell differentiation. In order to understand whether stem cell differentiation affects DNA damage repair, we compared DDRs and DNA repair in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) with their isogenic, differentiated progeny, including neural progenitor cells (neuroectodermal lineage) and their subsequent differentiation products: astrocytes and dopaminergic neurons. DNA damage repair by homologous recombination (HR) was significantly reduced after cell differentiation in all cells examined. Results Characterization of Differentiation Markers in iPSCs Human iPSCs (B12-2) and ESCs (H-9) were used to compare the DDR between undifferentiated and differentiated cell status. The cell lines used were positive for OCT4 or Nanog (Physique?1A) and cell markers (ectoderm -III tubulin [TUJ1], mesoderm clean muscle actin [SMA], and endoderm alpha-feto protein [AFP]) and confirmed for embryoid body (EB)-directed differentiation into the three germ layers. MCC950 sodium During EB-directed differentiation, the first germ layer to be formed is usually ectoderm, which is usually identified by the cell marker (TUJ1) in our temporal differentiation (d11). Further, from d14 onward, all three germ layers were observed as indicated (Physique?1B). In other words, on day 11 only TUJ1 stained well; SMA and AFP did not stain, MCC950 sodium which is reflected in the Physique?1B. Western blot analysis revealed a time-dependent decrease in Nanog, OCT4 (Physique?1C), and hMOF (Physique?1D), while sGC1 (Determine?1C) protein levels increased during differentiation. Levels of the hMOF acetylation product H4K16ac MCC950 sodium were also reduced in differentiated cells (Physique?1D) (Gupta et?al., 2008, Kumar et?al., 2011, Thomas et?al., 2008, Li et?al., 2012). During differentiation, levels of H4K20me2 and H3K9ac were not significantly reduced (Physique?1D). Open in a separate window Physique?1 Differentiation-Induced Changes in Stem Cell Markers and Histone Modifications (A) Immunostaining with antibody against Nanog and OCT4 in iPSCs. Scale bar, 10?m. (B) Immunostaining with different antibodies to detect stem cell differentiation into three germ layers. Scale bar, 10?m. (C and D) Western blot showing Nanog and OCT 4 and sGC 1 levels during various stages of differentiation (C) and western blot showing MOF, Histone H4, H4K16ac H3K9ac, Histone H3, and H4K20Me2 levels during temporal differentiation (D). Each experiment was done three independent occasions. NO Donors Induce Genomic Instability MCC950 sodium in Stem Cells We examined whether NO donors induced differentiation by treating stem cells with NOC-18 (5?M). Differentiation markers such as NKx2.5 (Figure?2A) and myosin light chain?2 (MLC2) protein (Physique?2B) were found to be significantly increased compared with controls. These results are consistent with our earlier report (Mujoo et?al., 2008). To determine whether NO also induces DDR, differentiated cells were treated with NOC-18 (0.5?mM) at a sub-toxic dose (95% survival), and response markers were analyzed by western blot and for Smad7 signaling/repair factor foci formation. Phosphorylation of ATM as well as that of Chk1, Chk2, and H2AX (Physique?2C) was detected.