The Key Laboratory of Pathobiology, Ministry of Education, Department of Pathology, College of Basic Medical Sciences, Jilin University, Changchun, 130021, Jilin, People's Republic of China.
Department of Pathology, The Affiliated Hospital of Qingdao University, Qingdao, 266000, Shandong, People's Republic of China.
Stem Cell Res Ther. 2020 Apr 16;11(1):157. doi: 10.1186/s13287-020-01664-0.
The development of somatic reprogramming, especially purely chemical reprogramming, has significantly advanced biological research. And chemical-induced extraembryonic endoderm-like (ciXEN) cells have been confirmed to be an indispensable intermediate stage of chemical reprogramming. They resemble extraembryonic endoderm (XEN) cells in terms of transcriptome, reprogramming potential, and developmental ability in vivo. However, the other characteristics of ciXEN cells and the effects of chemicals and bFGF on the in vitro culture of ciXEN cells have not been systematically reported.
Chemicals and bFGF in combination with Matrigel were used to induce the generation of ciXEN cells derived from mouse embryonic fibroblasts (MEFs). RNA sequencing was utilised to examine the transcriptome of ciXEN cells, and PCR/qPCR assays were performed to evaluate the mRNA levels of the genes involved in this study. Hepatic functions were investigated by periodic acid-Schiff staining and indocyanine green assay. Lactate production, ATP detection, and extracellular metabolic flux analysis were used to analyse the energy metabolism of ciXEN cells.
ciXEN cells expressed XEN-related genes, exhibited high proliferative capacity, had the ability to differentiate into visceral endoderm in vitro, and possessed the plasticity allowing for their differentiation into induced hepatocytes (iHeps). Additionally, the upregulated biological processes of ciXEN cells compared to those in MEFs focused on metabolism, but their energy production was independent of glycolysis. Furthermore, without the cocktail of chemicals and bFGF, which are indispensable for the generation of ciXEN cells, induced XEN (iXEN) cells remained the expression of XEN markers, the high proliferative capacity, and the plasticity to differentiate into iHeps in vitro.
ciXEN cells had high plasticity, and energy metabolism was reconstructed during chemical reprogramming, but it did not change from aerobic oxidation to glycolysis. And the cocktail of chemicals and bFGF were non-essential for the in vitro culture of ciXEN cells.
体细胞重编程的发展，尤其是纯化学重编程的发展，极大地推动了生物学研究的发展。化学诱导的类胚外内胚层（ciXEN）细胞已被证实是化学重编程不可缺少的中间阶段。它们在转录组、重编程潜力和体内发育能力方面与胚外内胚层（XEN）细胞相似。然而，ciXEN 细胞的其他特征以及化学物质和 bFGF 对 ciXEN 细胞体外培养的影响尚未得到系统报道。
采用化学物质和 bFGF 联合 Matrigel 诱导小鼠胚胎成纤维细胞（MEFs）生成 ciXEN 细胞。利用 RNA 测序检测 ciXEN 细胞的转录组，采用 PCR/qPCR 检测该研究相关基因的 mRNA 水平。采用过碘酸希夫染色和靛氰绿试验检测 ciXEN 细胞的肝向功能。通过乳酸检测、ATP 检测和细胞外代谢通量分析来分析 ciXEN 细胞的能量代谢。
ciXEN 细胞表达 XEN 相关基因，具有高增殖能力，能够在体外分化为内脏内胚层，并且具有分化为诱导性肝细胞（iHeps）的可塑性。此外，与 MEFs 相比，ciXEN 细胞上调的生物学过程主要集中在代谢方面，但它们的能量产生并不依赖于糖酵解。此外，在没有化学物质和 bFGF 鸡尾酒的情况下，ciXEN 细胞的生成必不可少，诱导性 XEN（iXEN）细胞仍然表达 XEN 标志物，具有高增殖能力和体外分化为 iHeps 的可塑性。
ciXEN 细胞具有高可塑性，在化学重编程过程中重建了能量代谢，但并没有从有氧氧化转变为糖酵解。并且化学物质和 bFGF 鸡尾酒对于 ciXEN 细胞的体外培养不是必需的。
