A pathological microenvironmental culture system consisting of cholestatic sera in duces embryonic stem cells to differentiate into hepatocyte-like cells in vitro / 中国病理生理杂志

ABSTRACT

AIM: To investigate whether a pathological micro-environmental culture system consisting of cholestatic sera induces embryonic stem cells (ESC) to differentiate into hepatocyte-like cells in vitro, and select hepatic stem cells from differentiating embryonic stem cells. METHODS: Mouse ESC, E14 cell line, were cultured in Dulbecco's modified Eagle's medium containing 106 U/L recombinant mouse leukemia inhibitory factor (rmLIF) and 10% FCS. After embryonic bodies formed by the hanging drop culture method, they were exposed to fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF) for one week, and then placed to a pathological micro-environmental culture system consisting of 5% cholestatic sera and cultured for 2 weeks. Morphological examination, immunocytochemical staining of albumin and CK8/18 were carried out, and mRNA level of albumin and transthyretin were detected by RT-PCR. Glycogen storage and urea synthesis of the cells were tested with PAS staining and colorimetric assay, respectively. RESULTS: The proliferation of cells was inhibited at the early stage when cultured in a pathological micro-environmental culture system consisting of 5% cholestatic sera, but 2 weeks later, a large number of epithelial-like cell colonies were observed, which exhibited hepatocellular phenotype, expressing albumin and CK8/18, transcribing mRNA of albumin and transthyretin and synthesizing glycogen and urea. CONCLUSION: A pathological micro- environmental culture system consisting of 5% cholestatic sera could not only induce embryonic stem cells to differentiate into hepatocyte-like cells, but select hepatic stem cells from differentiating embryonic stem cells initially induced by FGF-4 and HGF in vitro as well.