Improved Differentiation of Mesenchymal Stem Cells into Hepatocyte-like Cells using FGF4 and IGF-1 in 3D Culture

Human Umbilical Cord Mesenchymal Stem Cells (UCMSCs) are considered as an excellent candidate for cell therapy to treat end-stage liver disease. Fibroblast Growth Factor-4 (FGF4), Hepatocyte Growth Factor, and Insulin-like Growth Factor-1 are some of the critical cytokines involved in liver development and regeneration. To evaluate the differentiation potency of cells into hepatocyte-like cells we used these cytokines. UCMSCs were isolated from Wharton's jelly of fullterm infants. The cells were characterized as MSCs by flow-cytometry and their multilineage differentiation capacity. Then, UCMSCs were cultured in 3D collagen scaffold and hepatogenic media with or without FGF4 for 21 days and the data were compared to control. The expression of liver specific genes was evaluated by real-time quantitative RT-PCR and immunocytochemistry. These cells expressed MSC markers and could differentiate into adipocytes and osteocytes. A non?significant higher level of liver specific genes, such as cytokeratin-18 and 19, alpha-fetoprotein and albumin, and also a significant higher level of CYP2B6 expressed by UCMSCs in hepatogenic medium containing FGF4 compared with control. In some specimens, cytokeratin-19-positive cells surrounded a luminal space within collagen scaffolds. Liver-specific marker expression was increased by pre-exposing the cells to FGF4 before treating with IGF-1 and HGF in 3D collagen scaffold. Keywords: Umbilical Cord Mesenchymal Stem Cells; 3D Culture; Collagen Scaffold; Fibroblast Growth Factor-4; Hepatocyte-Like Cell; Insulin-Like Growth Factor-1. Abbreviations: UCMSCs: Human Umbilical Cord Mesenchymal Stem Cells; FGF4: Fibroblast Growth Factor 4; HGF: Hepatocyte Growth Factor; IGF-1: Insulin-like Growth Factor-1; MSCs: Mesenchymal Stem Cells; ICG: Indocyanine green; PAS: periodic acid Schiff; CK-18: cytokeratin-18; CK-19: Cytokeratin-19; AFP: alpha-fetoprotein; G6P: glucose 6 phosphatase; PEPCK: phosphoenolpyruvate carboxykinase; TAT: tyrosine amino transferase; FBS: Fetal Bovine Serum; OSM: oncostatin M; RT-PCR: Reverse Transcription Polymerase Chain Reaction; PBS: Phosphate-Buffered Saline; Hep- Par1: Hepatocyte paraffin 1; DAB: Diaminobenzidine; CYP2B6: Cytochrome P450 2B6.