The Establishment Of Sequential Inducible Reprogramming System And The Identification Of Pluripotent Marker Gene

Somatic cells can be converted into induced pluripotent stem cells(iPSCs) by lentiviral transduction of four embryonic stem cell(ESC)-expressed transcription factors:Oct4,Sox2,Klf4, and c-Myc. It is one of the notable landmarks in the progress of regenerative medicine, which could be used to construct disease models and to screen effective and safe drugs, as well as to treat patients through cell transplantation therapy.iPS cells are turely pluripotent via tetraploid complementation experiment. It means iPSCs can differentiate any type of somatic cells. Somatic cell nuclear transfer (SCNT) and induction of pluripotency by defined transcription factor are two major approaches that can reprogram differentiated mammalian somatic cells to pluripotent state, and even topitent state by SCNT. Results from a most recent study have demonstrated that mice can be successfully re-cloned to more than twenty-five generations through serial nuclear transfer. Therefore, it is worth to explore how many generations the all-iPSC mice can be serially produced using iPS system. Using a sequential reprogramming approach based on the doxycycline (Dox)-inducible iPS system, The10-all-iPSC mice were generated from one Tet-on inducible iPSC line that was initially derived from mouse embryonic fibroblasts (MEF). Subsequently, somatic cells were retrieved from the tissue of the10-all-iPSC mice. After the addition of Dox to the induction medium,20-iPSC colonies emerged and viable fertile all-iPSC mice could be generated from the20-iPSC lines through tetraaploid complementation. We then established30-,40-,50-, and60-all-iPSC lines from somatic cells retrived from the previous generation of the all-iPSC mice, and30-,40-,50-, and60-all-iPSC mice were subsequently produced through tetraaploid complementation.Based on the sequential reprogrammed system, we utilized genetically well-defined iPSCs combined with the genome-wide levels of mRNA expression, small RNA profile, core histone modifications (H3K27me3, H3K4me3, and H3K4me2), and DNA methylation high throughput sequencing to characterize the genome-wide differences between4N-ON and4N-OFF iPS cell lines. The study demonstrates that iPSCs are globally similar at the levels of mRNA, small RNA, core hsitone modification and DNA melthylaiton. Most importantly,we have discovered that the consistent loss of DNA methylation of Zrsrl correlated with the reduced pluripotency of iPSCs derived by different combination of transcription factors.Moreover, in this study, to find an alternative source of high-quality iPSCs, we investigated the reprogramming of HSC/HPC at the molecular level. Our study demonstrates that HSC/HPC are amenable to repprgramming into a high frequency of high-quality iPSCs with full pluripotency and unbiased hematopoietic differentiation potential. By genome-wide analyses, we found that HSC/HPC are amenable to reprogramming, including the down-regulation of mesenchymal genes and up-regrulation of cell cycle-accelerating genes. In conclusion, HSC/HPC-iPSCs recapitulate ESCs in term of pluripotency and differentiation potential, which supports the preclinical biosafety of iPSCs.In summary, the study demonstrated for the first time that sequentially reprogrammed iPSCs could produce all-iPSC mice for up to six generations. Subsequently, based on the system,we revealed that loss of methylation of Zrsrl correlated with the reduced pluripotency of iPSCs. Moreover, the findings showed that HPC are an alternative source for getting high-quality iPSCs. These studies would improve the quality of iPSCs and shed light on the clinical of iPSCs.