Laboratory of Stem Cells and Translational Medicine, Institutes for Life Sciences, School of Medicine, South China University of Technology, Guangzhou 510006, China.
School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China.
Cells. 2023 Feb 2;12(3):497. doi: 10.3390/cells12030497.
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/EBPβ 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/EBPβ is directly bound to the promoter region of proliferating genes to promote cell proliferation; this interaction between C/EBPβ and DNA sequences of the promoters was verified by luciferase assay. On the contrary, the knockdown of C/EBPβ 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/EBPβ. Moreover, the results of CUT&Tag sequencing showed that C/EBPβ also directly bound the promoter of E2F2, regulating E2F2 expression. Interestingly, Co-IP analysis exhibited a direct binding between C/EBPβ 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/EBPβ 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.
人胚胎干细胞（hESCs）具有解决临床应用和药物开发中功能性肝细胞短缺问题的潜力。然而，由于其增殖能力有限，大量可用于的 hESC 来源的肝细胞不能有效获得。在这项研究中，我们发现肝转录因子 C/EBPβ 在肝分化过程中的增强不仅可以显著促进肝基因的表达，如白蛋白、甲胎蛋白和α-1 抗胰蛋白酶，还可以显著增强增殖相关表型，包括增加增殖基因的表达，如 CDC25C、CDC45L 和 PCNA，以及细胞周期和 DNA 复制途径的激活。此外，CUT&Tag 测序分析进一步表明，C/EBPβ 直接结合到增殖基因的启动子区域以促进细胞增殖；通过荧光素酶测定验证了 C/EBPβ 与启动子 DNA 序列之间的这种相互作用。相反，C/EBPβ 的敲低可以显著抑制上述增殖基因的表达。RNA 转录组分析和 GSEA 富集表明 E2F 家族富集，并且随着 C/EBPβ 的过表达或敲低，E2F2 的表达发生变化。此外，CUT&Tag 测序结果表明 C/EBPβ 还直接结合到 E2F2 的启动子上，调节 E2F2 的表达。有趣的是，Co-IP 分析显示 C/EBPβ 与 E2F2 蛋白之间存在直接结合，并且在 LO2 细胞系（一种肝祖细胞系）中也验证了这两种蛋白之间的这种相互作用。因此，我们的结果表明 C/EBPβ 首先启动 E2F2 的表达，然后与 E2F2 结合，在 hESC 的分化过程中调节肝细胞中增殖基因的表达。因此，我们的发现为提供一种体外高效生成增殖性肝细胞的方法开辟了新途径，可能满足细胞治疗以及药物和毒理学研究中对其的需求。
