The Role Of Base Excision Repair In Cellular Reprogramming

Stem cells are a kind of cells with two properties: self-renewal and pluripotency.The most representative two of them are embryonic stem cells and induced pluripotent stem cells(iPSCs).Embryonic stem cells are derived from the inner cell mass(ICM)of blastocysts and can differentiate into all embryonic cell types except the placenta.Embryonic stem cells are considered to hold great application prospects in the fields of regenerative medicine,drug screening,pharmacotoxicology and developmental biology research due to their properties.However,the use of human embryonic stem cells is controversial because of ethical and moral issues.iPSCs are dedifferentiated from terminal somatic cells through ectopic expressing reprogramming factors.The team led by Shinya Yamanaka first reported the method to get iPSCs in 2006.iPSCs are quite similar to embryonic stem cells in every way.The high-quality iPSCs after screening can differentiate into all cell types of an animal.The derivation of iPSCs does not require human embryos,which avoids the debate on ethics and morals.Moreover,immunological rejection after cells/tissue transplantation can be greatly reduced because of the same genetic background of donor iPSCs and recipient patients.Above all,iPSCs have been considered as an excellent alternative to embryonic stem cells in the fields of clinical application and basic research.However,there are still several challenges,such as low induction efficiency of reprogramming and cellular heterogeneity,genomic instability,and tumorigenicity of iPSCs,that need to be solved.Genome integrity is quite important because iPSCs will divide and differentiate into a large number of different types of cells.But the reprogramming process would introduce thousands of genetic aberrations,which greatly impair the genome integrity of iPSCs and further affect their safety in clinical applications.Therefore,how to preserve genome stability throughout the reprogramming process is a critical issue to be solved.In 2015,scientists in our laboratory established the six-generation sequential reprogramming system and found that single nucleotide variants(SNV)were generated and gradually accumulated during the reprogramming process,which in turn impaired the potency of iPSCs.In this study,we used the well-established reporter systems to detect the efficiency of 4 DNA damage repair pathways including nucleotide excision repair(NER),base excision repair(BER),homologous recombination(HR),and nonhomologous end joining(NHEJ)in the sequential reprogramming system and the classical 4-factor reprogramming process.The results showed that BER is crucial for the quality of iPSCs,that is high-quality iPSCs usually have stronger BER efficiency.And we found that the BER efficiency is insufficient to cope with the rapidly rising reactive oxygen species(ROS)in the early and middle stages of the reprogramming process,unable to cope with the rapidly rising reactive oxygen species(ROS)during the reprogramming process(ROS)and their resulting oxidative damage,which further led to the untimely repair of DNA damage.We chose 6 key factors involved in BER including Ogg1,Apex1,Parp1,Xrcc1,Lig3,and Polβ through literature and experience.By overexpressing each of them,we found that overexpression of Xrcc1 could increase the efficiency of BER efficiency,which in turn improves the potency of iPSCs.Overexpressing Xrcc1 during classical 4-factor reprogramming significantly enhanced the BER,which further reduced the SNV in the resultant iPSCs and eliminated the reported mutational signature SBS17 b induced by the reprogramming process.Meanwhile,this Xrcc1-mediated BER enhancement also improved the reprogramming efficiency and the potency of iPSCs.In conclusion,our study presents a feasible method that can improve the genomic stability and pluripotency of iPSCs,and this provides a new idea for the safe clinical applications of iPSCs.