C/EBP beta Coupled with E2F2 Promoted the Proliferation of hESC-Derived Hepatocytes through Direct Binding to the Promoter Regions of Cell-Cycle-Related Genes

卷:12

Article

关键词: EMBRYONIC STEM-CELLS;    FUNCTIONAL HEPATOCYTES;    EFFICIENT GENERATION;    LIVER-FUNCTION;    DIFFERENTIATION;    PROTEIN;    EXPANSION;    SYSTEMS;   

DOI  :  10.3390/cells12030497

来源: SCIE

【 摘 要 】

Human embryonic stem cells (hESCs) hold the potential to solve the problem of the shortage of functional hepatocytes in clinical applications and drug development. However, a large number of usable hepatocytes derived from hESCs cannot be effectively obtained due to the limited proliferation capacity. In this study, we found that enhancement of liver transcription factor C/EBP beta during hepatic differentiation could not only significantly promote the expression of hepatic genes, such as albumin, alpha fetoprotein, and alpha-1 antitrypsin, but also dramatically reinforce proliferation-related phenotypes, including increasing the expression of proliferative genes, such as CDC25C, CDC45L, and PCNA, and the activation of cell cycle and DNA replication pathways. In addition, the analysis of CUT & Tag sequencing further revealed that C/EBP beta is directly bound to the promoter region of proliferating genes to promote cell proliferation; this interaction between C/EBP beta and DNA sequences of the promoters was verified by luciferase assay. On the contrary, the knockdown of C/EBP beta could significantly inhibit the expression of the aforementioned proliferative genes. RNA transcriptome analysis and GSEA enrichment indicated that the E2F family was enriched, and the expression of E2F2 was changed with the overexpression or knockdown of C/EBP beta. Moreover, the results of CUT & Tag sequencing showed that C/EBP beta also directly bound the promoter of E2F2, regulating E2F2 expression. Interestingly, Co-IP analysis exhibited a direct binding between C/EBP beta and E2F2 proteins, and this interaction between these two proteins was also verified in the LO2 cell line, a hepatic progenitor cell line. Thus, our results demonstrated that C/EBP beta first initiated E2F2 expression and then coupled with E2F2 to regulate the expression of proliferative genes in hepatocytes during the differentiation of hESCs. Therefore, our findings open a new avenue to provide an in vitro efficient approach to generate proliferative hepatocytes to potentially meet the demands for use in cell-based therapeutics as well as for pharmaceutical and toxicological studies.