Abstract
Abstract
Background
Induced hepatic stem cells (iHepSCs) with the capacities of self-renewal and bidifferentiation into hepatocytes and cholangiocytes were generated from mouse embryonic fibroblasts (MEFs) by lineage reprogramming in our previous research. However, the mechanism of iHepSC self-renewal has not been elucidated. Active demethylation regulated by Tet1 plays an important role in the self-renewal of stem cells, including pluripotent stem cells and adult stem cells. Here, we investigated the role and mechanism of Tet1-regulated demethylation in the self-renewal of iHepSCs.
Methods
The methylation levels and the expression of Tet1 in iHepSCs and MEFs were analyzed by immunofluorescent staining, quantitative reverse transcription PCR and western blotting. Then, the effects of Tet1 knockdown on the proliferation and self-renewal of iHepSCs were analyzed by CCK8, colony formation, and sphere formation assays. The mechanism by which Tet1 regulates the self-renewal of iHepSCs was investigated by chromatin immunoprecipitation, bisulfite sequence PCR, and methylation-sensitive restriction endonuclease-PCR.
Results
The high level of 5hmC and the low level of 5mC in iHepSCs were accompanied by high expression of Tet1. After Tet1 expression was knocked down by shRNA in iHepSCs, the proliferation and self-renewal capacities were inhibited, and the expression of Myc was also decreased. The higher expression level of Myc in iHepSCs maintained its self-renewal and was regulated by Tet1, which directly binds to CBS-1 and site A regions of the Myc promoter and demethylates the CpG cytosine. In addition, CTCF also binds to the CBS-1 and site A regions of the Myc promoter and regulates Myc expression along with TET1.
Conclusion
The self-renewal of iHepSCs was maintained by the higher expression of Myc, which was coregulated by TET1 and CTCF. This study may provide new insights into the self-renewal of stem cells, which can promote the research and application of ‘reprogrammed’ stem cells.
Funder
National Natural Science Foundation of China
Foundation strengthening program in technical field of China
Publisher
Springer Science and Business Media LLC
Subject
General Biochemistry, Genetics and Molecular Biology
Reference49 articles.
1. Horisawa K, Suzuki A. Direct cell-fate conversion of somatic cells: toward regenerative medicine and industries. Proc Jpn Acad Ser B Phys Biol Sci. 2020;96(4):131–58.
2. Yu B, He ZY, You P, Han QW, Xiang D, Chen F, et al. Reprogramming fibroblasts into bipotential hepatic stem cells by defined factors. Cell Stem Cell. 2013;13(3):328–40.
3. Yu B, Li H, Chen J, He Z, Sun H, Yang G, et al. Extensively expanded murine-induced hepatic stem cells maintain high-efficient hepatic differentiation potential for repopulation of injured livers. Liver Int. 2020;40(9):2293–304.
4. He S, Nakada D, Morrison SJ. Mechanisms of stem cell self-renewal. Annu Rev Cell Dev Biol. 2009;25:377–406.
5. Li Y, Chen X, Lu C. The interplay between DNA and histone methylation: molecular mechanisms and disease implications. EMBO Rep. 2021;22(5):e51803.