Efficient exogenous DNA-free reprogramming with suicide gene vectors

Abstract

Reprogramming with episomal vectors is an easy, safe, and cost-effective method to generate exogenous DNA-free (exogene-free) induced pluripotent stem cells (iPSCs). However, the genomic integration of exogenes is observed occasionally. Additionally, the removal of episomal DNA takes more than 70 days in established iPSCs. Here, we inserted the cytosine deaminase (CD) gene from yeast into episomal vectors and used them to reprogram human fibroblasts into iPSCs. These new episomal vectors (CD episomal vectors) were eliminated from the generated iPSCs as early as seven days after 5-fluorocytosine (5-FC) treatment. We also found that cells with the integration of the CD gene perished within two days of 5-FC treatment. In addition, we generated exogene-free induced neural stem cells after one passage by direct reprogramming with CD episomal vectors combined with 5-FC treatment. Conclusively, our novel method allows the rapid and easy isolation of exogene-free reprogrammed cells and can be applied to disease modeling and clinical applications.