Experimental study on the induction of human hepatocytes into fibroblasts in vitro by nuclear factor 4α combined with histone deacetylase inhibitor and DNA methyltransferase inhibitor

Abstract

Objective To induce the hepatocellular transdifferentiation by transfecting human hepatocytes with nuclear factor 4α (HNF4A) and performing epigenetic modification [histone deacetylase inhibitor (HDACi) and DNA methyltransferase inhibitor (DNMTi)], aimed at establishing a brand new and more efficient liver cell direct conversion method. Methods The human foreskin fibroblasts (hFFs) of 8th generation were induced reprogramming by culturing in the presence of 5-azacytidine (5-AzaC) and valproic acid (VPA). Three days later, the cells were infected with HNF4A lentivirus and then continuously cultured in the hepatocyte culture medium for 20 days. Finally, The dynamic process of morphological changes was observed by inverted phase contrast microscope. The specific hepatocellular genes were detected by real-time quantitative polymerase chain reaction (Real-time PCR), and the hepatocellular biological function was proven by performing indocyanine green (ICG) uptake test, periodic acid-schiff (PAS) staining and assay of urea synthesis. Results During the induction, the cells gradually changed from fusiform to epithelioid in morphology, and gradually began to overexpress liver specific gene and perform biological function. Twenty days after transdifferentiation, the hepatocyte-like cells exhibited significant hepatocellular epithelial cells change in morphology with abundant cytoplasm, large circular stained nucleus and prominent nucleoli. Real-time PCR detection indicated that most liver-specific genes were upregulated, especially albumin (ALB) and transferrin (TF), with the difference being statistically significant (t=7.418, 5.131, P=0.002, 0.001). In ICG uptake test, PAS staining and assay of urea synthesis, the hepatocyte-like cells revealed more hepatocellular biological functional maturation than normal fibroblasts, and a lot of them was close to the levels of normal hepatocytes in biological function with the difference being statistically significant (P=0.031, 0.013). Conclusion Overexpression of HNF4A and performing epigenetic modification (HDACi and DNMTi) jointly promote the differentiation of human skin fibroblasts into hepatocyte-like cells, as assessed by morphological, genetic and functional tests. Key words: Hepatocyte; Transdifferentiation; Human hepatocyte nuclear factor 4α; Histone deacetylation; DNA methylation