Hepatic differentiation of rat induced pluripotent stem cells in vitro.

Abstract

To show the efficient generation of hepatocyte-like cells (HLCs) differentiated from the induced pluripotent stem cells (iPSCs) of rats. Hepatic differentiation was achieved using a three-step protocol with several growth factors. First, rat iPSCs were differentiated into definitive endoderm cells using Activin A and Wnt3a treatment. Then fibroblast growth factor 4 and bone morphogenetic protein 2 were added to the culture medium and used to induce hepatic differentiation. Finally, hepatocyte growth factor, Oncostatin M and dexamethasone were used for hepatic maturation. The liver-related markers and functions of HLCs were assessed at the gene and protein levels. After endodermal induction, the differentiated cells expressed endodermal markers forkhead box protein A2 and SRY-box containing gene 17 at the mRNA and protein levels. After 20 d of culture, the iPSCs were differentiated into HLCs. These differentiated cells expressed hepatic markers including α-fetoprotein, albumin CK8, CK18, CK19, and transcription factor HNF-4α. In addition, the cells expressed functional proteins such as α1-antitrypsin, cytochrome P450 1A2 and CYP 3A4. They acted like healthy hepatic cells, storing glycogen and taking up indocyanine green and low-density lipoproteins. Also, the rates of urea synthesis (20 d 1.202 ± 0.080 mg/dL vs 0 d 0.317 ± 0.021 mg/dL, P < 0.01) and albumin secretion (20 d 1.601 ± 0.102 mg/dL vs 0 d 0.313 ± 0.015 mg/dL, P < 0.01) increased significantly as differentiation progressed. Rat iPSCs can differentiate into HLCs rapidly and efficiently. These differentiated cells may be an attractive resource for treatment of end-stage liver disease.