While four transcription factors mediated reprogramming could potentially induce or enlarge mutations in the iPSCs, also the impacts of the mutations on the developmental potential of the iPSCs need further evaluation. Based on the a sequential reprogramming system combining with whole genome sequencing, the lab of Prof. CAI Jun from Beijing Institute of Genomics, Chinese Academy of Sciences , in collaboration with Prof. GAO shaorong from the School of Life Sciences and Technology, Tongji University and Prof. TIAN Jianhui from College of Animal Sciences and Technology, China Agricultural University, revealed accumulated single-nucleotide variations (SNVs) throughout the sequential reprogramming process, which accounted for the gradual decrease in viability of the all-iPSC mice. The work has been published in Nature Communications on February 18, 2015.

In this sequential reprogramming system, the viabilities of later-generation all-iPSCs were greatly reduced. Combined MeDIP-seq and RNA-seq results indicated the decreased viability was unrelated with epigenetic effects. Further investigation revealed amount SNVs accumulated throughout the sequential process, and functional annotation of these SNVs demonstrated that these mutations would cause developmental failures in mice, also corresponding abnormal phenotypes were supported by histopathological examination. The origins of these accumulated SNVs were evaluated via droplet digital PCR (ddPCR), and results showed that about two-thirds of the SNVs pre-existed in the all-iPSC mouse tissues which generated during development, rather than merely in iPSC induction.

Another interesting phenomenon in this study is the recurrent loss of retrotransposon in iPSCs, and same pattern occurred in other pluripotent stem cells, including ESCs, ntESCs and blastocyst. Relatively, the deleted retrotransposons were regained in differentiated cells after differentiation either in vitro and vivo. However, why unique loss of retrotransposon in pluripotent stem cells deserved further investigations and concerns.

This study provides information to better understand the association between gene mutations and developmental effect, which is essential for screening pre-clinical bio-safety iPSCs.

The accumulated SNVs and their frequencies during sequential reprogramming（Image by CAI Jun’s group）