Characterization and in vitro differentiation of iPS cells to investigate DNA glycosylase Neil3 | Characterization and in vitro differentiation of iPS cells to investigate DNA glycosylase Neil3

DNA glycosylases play an important role in DNA repair, keeping at bay the accumulation of DNA damage. Neil3 has been shown to be a functional DNA glycosylase in vitro with a proliferative tissue-specific expression, highly expressed in stem cells. Yet, its biological function remains largely unknown. In order to understand the role of Neil3 and illuminate the effects of DNA damage in stem cells, we used iPS cells previously generated in this lab from wild type and Neil3-deficient mice. We characterized the stem cell properties of our iPS cells and began to establish differentiation and characterization of differentiation capacity. The neil3-/- iPS cells showed increased levels of the stem cell markers alkaline phosphatase, Rex1 and Klf4. Differentiation experiments showed successful embryoid body formation and neural differentiation but also revealed the need for homogeneity during initial embryoid body formation. Oxidant and antioxidant treatment indicated that DNA damage accumulates differently in wild-type and neil3-/- iPS cells and morphology studies revealed effects of treatment on differentiation. Our results indicate that neil3-/- iPS cells have a higher propensity for the pluripotent state and may have defects in differentiation. We speculate that Neil3 may play a role in maintaining DNA integrity in iPS cells and is required for appropriate differentiation.

DNA glycosylases play an important role in DNA repair, keeping at bay the accumulation of DNA damage. Neil3 has been shown to be a functional DNA glycosylase in vitro with a proliferative tissue-specific expression, highly expressed in stem cells. Yet, its biological function remains largely unknown. In order to understand the role of Neil3 and illuminate the effects of DNA damage in stem cells, we used iPS cells previously generated in this lab from wild type and Neil3-deficient mice. We characterized the stem cell properties of our iPS cells and began to establish differentiation and characterization of differentiation capacity. The neil3-/- iPS cells showed increased levels of the stem cell markers alkaline phosphatase, Rex1 and Klf4. Differentiation experiments showed successful embryoid body formation and neural differentiation but also revealed the need for homogeneity during initial embryoid body formation. Oxidant and antioxidant treatment indicated that DNA damage accumulates differently in wild-type and neil3-/- iPS cells and morphology studies revealed effects of treatment on differentiation. Our results indicate that neil3-/- iPS cells have a higher propensity for the pluripotent state and may have defects in differentiation. We speculate that Neil3 may play a role in maintaining DNA integrity in iPS cells and is required for appropriate differentiation.