Valproic Acid-Induced Hepatocyte-Like Differentiation of Human Amniotic Epithelial Stem Cells in 2D Microenvironment

Abstract

This experimental in-vitro study assessed the effectiveness of the 21-day differentiation of human amniotic epithelial cells (hAECs) into hepatic-like cells (HLC), displaying hepatocytes with functional features using culture media supplemented with Valproic Acid (VPA). hAECs were isolated from the human placenta which was taken from patients with full-term pregnancy after C-sections. The differentiated cells were analyzed for hepatic markers by qPCR, PAS staining for glycogen storage & Immunocytochemistry. The results demonstrated that the hAECs with large nuclei, sparse cytoplasm, and cobblestone-like structure morphology changed into single cells characterized by a polygonal shape with abundant cytoplasm, and having huge bright vacuoles resembling fat droplets after being treated with 2.5mM concentration of VPA for 21 days. These cells exhibited substantial upregulation of hepatic gene expression, CYP3A4, CYP27B1, Alpha-fetoprotein (AFP), Albumin, HNF1A, HNF3B, and hepatic protein, Albumin, signifying that hAECs possessed normal functioning hepatocyte characteristics. Analysis of H3 and H4 by immunofluorescence showed an increase in VPA-treated cells indicating alteration in the histone acetylation of the cells. Further analysis showed enhanced glycogen storage in the differentiated cells, a significant functional characteristic of hepatocytes. This research identified an appropriate substrate for the differentiation of hAECs into functional hepatocytes, their successful therapeutic application would represent an advancement towards the development of tissue-engineered liver tissue.