Production of Hepatocytes Derived from Chicken Embryos Across Three Developmental Stages

Abstract Background: Chicken hepatocytes provide a valuable resource for conducting cell-based assays and gaining insights into the underlying mechanisms of diseases. Objective: In this study, we examined in vitro generation of chicken hepatocytes from three developmental stages. Methods: In this in vitro study , chicken embryos aged 5, 7, and 10 days were used. The proliferation of chicken hepatocytes was assessed in DMEM/F12+10% FBS medium. After 3 days, the proliferation rate of hepatocytes, hepatocytes-specific gene expression, hepatocytes-specific enzymes (ALT and AST), albumin production and urea secretion were assessed. Findings: Morphological examination of individual hepatocytes exhibited a characteristic hexagonal structure with prominent nuclei and nucleoli, surrounded by a marginal cytoplasm. E10 embryos exhibiting a markedly higher proliferation rate in comparison to those from E5 and E7 stages. In contrast to E10, approximately 50% of hepatocytes derived from E5 and E7 embryos exhibit reduced proliferation after 3 days. In the suspension culture, hepatocytes had the ability to form spheroids or hepatospheres. The expression of hepatocyte-specific genes such as AFP, ALP, FOXA2, and CYP3A4 was markedly higher in E10-derived hepatocytes compared to E5-derived hepatocytes. Conversely, the expression of CXCR4, OCT4, NANOG, and SOX17 was significantly lower in E10-derived hepatocytes than in E5-derived hepatocytes. There was a significant increase in the expression of ALT and AST in E10-derived hepatocytes compared to E5-derived hepatocytes. Furthermore, Albumin production was significantly lower in E10-derived hepatocytes compared to E5-derived hepatocytes. While, urea secretion was higher in E10-derived hepatocytes compared to E5-derived hepatocytes Conclusion : Based on our findings, we have determined that E10 represents an optimal stage for the derivation and proliferation of chicken hepatocytes in vitro.