Antioxidant Treatment During Reprogramming Reduces Genomic Aberrations Of Human IPSCs

BackgroundHuman embryonic stem (ES) cells can proliferate indefinitely and differentiate into various types of cells in vitro, which can theoretically provide sufficient number of cells for replacement therapy in regenerative medicine. However, the application potential of Human ES cells is restricted due to the ethical issues associated with the derivation. In2006, Shinya Yamanaka’s group reported that forced expression of4transcription factors (Oct3/4, SOX2, Klf4, c-Myc) reprogram murine cells to pluripotent cells with properties similar to ESCs, termed Induced Pluripotent Stem Cells (iPSCs). Reprogramming technology bypassed the ethical issues associated with ESCs, and thus holds great promise for regenerative medicine, drug screening and disease modeling etc. However, mounting studies have reported that Human iPSCs possess genomic aberrations, which poses safety concerns and may impede the applications in regenerative medicine. Thus, identification of aspects causing genomic aberrations and development of methods to protect the genome integrity of Human iPSCs are important to generate safer iPSCs for potential clinical application. In this thesis, our group demonstrated that treatment with antioxidant in the process of reprogramming, can lead to the reduction of genomic instability. In addition, iPS cells cultured with antioxidants have the less copy number variation. Our results show that antioxidants supplementation can enhance the quality and safety of Human iPSCs in the process of inducing and maintaining iPSCs, so it provides vital value for IPS’s safety and wide medicine application. And iPS cells are expected to become an important source of cells for regenerative medicine and cell therapy. This study is divided into two parts:1) The establishment of IPS system;2) Antioxidant supplementation reduces genomic aberrations in Human induced pluripotent stem cellsPart I The establishment of Human reprogramming SystemObjective:To establish the generation of iPSCs from Human foreskin fibroblasts (HFFs) and urine cells.Methods:We used the PMX retroviruses carrying Human OCT4, SOX2, KLF4, c-MYC to infect the urine cell/Human fibroblast cells.Results:Human foreskin fibroblasts and urine cells can be reprogrammed into ALP positive iPSCs.Conclusion:Urine cells are more easily reprogrammed into iPSCs than HFFs.Part Ⅱ Antioxidant Supplementation Reduces Genomic Aberrations in Human Induced Pluripotent Stem CellsObjective:Human iPSCs harbor genomic aberrations, suggesting that reprogramming may be mutagenic. In this study, we aimed to examine genotoxicity caused by oxidative stress during reprogramming and develop strategies to mitigate oxidative stress to generate high-quality human iPSCs.Methods:We measured ROS level by flow cytometry and DNA damage by y-H2AX staining during reprogramming. We then supplemented the Medium with antioxidants during reprogramming and examined the copy number variations (CNVs) of the resultant iPSCs genome by microarray. Results:Antioxidant supplementation mitigated oxidative genotoxicity during reprogramming. Compared with iPSCs made without antioxidants,iPSCs added with antioxidants had fewer de novo copy number variations.Conclusion:Antioxidant supplementation can reduce genomic instabilities in human iPSCs.