Xrcc1 improves the genetic stability and pluripotency of iPSCs by enhancing BER

This study is led by Shaorong Gao (the Dean of School of Life Sciences and Technology and the Director of the Frontier Science Center for Stem Cell Research, Tongji University), Shuai Gao (National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University), Prof. Zhiyong Mao (Shanghai Key Laboratory of Maternal Fetal Medicine, Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, Frontier Science Center for Stem Cell Research, Tongji University).

Induced pluripotent stem cells (iPSCs) hold great promise for broad clinical application due to their extraordinary ability to self-renew and differentiate into different functional cell types. However, many studies have shown that the process of reprogramming introduces a number of genetic aberrations represented by single-nucleotide variants (SNVs) and copy number variations (CNVs), raising safety concerns for clinical applications

This team previously demonstrated that genetic alterations caused by accumulated SNVs impaired the development potential of resultant iPSCs using a sequential reprogramming approach based on the doxycycline (Dox)-inducible iPSC system. But the reasons for the generation and accumulation of SNVs have yet to be fully elucidated.

The base excision repair (BER) pathway is responsible for coping with DNA damage derived from deamination, including SNVs. To determine which DNA repair pathway plays a vital role during cellular reprogramming, this study first tested the efficiency of BER pathways during the reprogramming of secondary doxycycline (dox)-inducible mouse embryonic fibroblasts (2⁰-MEFs). The findings indicated that BER plays an essential role in cellular reprogramming. Additionally, the insufficient BER efficiency in differentiated cells brought increased DNA lesions that were reflected by the elongation of the comet assay tail moment and increased accumulation of SNVs from the early and middle stages of reprogramming.

Here, they reported a more universal and effective method to reduce SNVs during reprogramming by enhancing the BER ability through Xrcc1.

They found that Xrcc1-overexpressed iPSCs exhibited remarkably high resistance to H₂O₂, which can induce severe oxidative DNA damage. Next, the knockdown of Xrcc1 with shRNA reduced the efficiency of BER in iPSCs, which proved the necessity of Xrcc1 for BER. In conclusion, we can efficiently enhance or reduce BER by regulating the expression of Xrcc1. Therefore, using Xrcc1 to enhance the BER’s ability to address increased oxidative DNA damage in the early stage of reprogramming may be able to improve the efficiency of iPSC generation.

This study then demonstrated that the enhancement of BER can boost the efficiency of reprogramming, which may be attributed to the decrease in apoptotic cells in the early stage of reprogramming. And they found that BER is an important participant in the cellular reprogramming process, while BER ability in the early stage is insufficient and can be improved by Xrcc1.

The team also determined whether the enhanced BER ability improves the genetic stability of the resultant iPSCs. The results indicated that the enhanced BER by Xrcc1 helps cells protect against rapidly increasing ROS pressure and efficiently preserves genome integrity during cellular reprogramming. The analysis of the mutational signature further implied that the augmented BER efficiency during the reprogramming process was sufficient to promote genome integrity in the resultant iPSCs.

Finally, the chimera assay, an important criterion for high-quality iPSCs, was used to further evaluate the development potential of resultant iPSCs. And the data demonstrated that the quality of iPSCs was markedly improved by Xrcc1-mediated enhancement of BER during reprogramming.

Taken together, with their previous observation that SNVs gradually accumulated over the sequentially reprogramming generations, this study found that BER efficiency is insufficient in the early stage of reprogramming to cope with the rapidly raised reactive oxygen species (ROS) stress. This resulted in a large amount of oxidative damage that could not be repaired in time. Further efforts successfully identified that Xrcc1 overexpression can enhance BER during reprogramming. Next, they found that the Xrcc1-mediated enhancement of BER ability improved the reprogramming efficiency and greatly reduced the mutations of resultant iPSCs, which further improved their quality. These results indicate that Xrcc1 is a potential detectable and application target in screening high-quality iPSCs.

See the article:

Enhancement of Xrcc1-mediated base excision repair improves the genetic stability and pluripotency of iPSCs

https://doi.org/10.1016/j.scib.2022.04.003

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