Abstract
Hepatocyte transplantation is considered to be a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) provide an unlimited source for the generation of functional hepatocytes. In this study, we generated iPSCs from porcine ear fibroblasts (PEFs) by overexpressing Sox2, Klf4, Oct4, and c-Myc (SKOM), and developed a novel strategy for the efficient differentiation of hepatocyte-like cells from porcine iPSCs by following the processes of early liver development. The differentiated cells displayed the phenotypes of hepatocytes, exhibited classic hepatocyte-associated bio-functions, such as LDL uptake, glycogen storage and urea secretion, as well as possessed the metabolic activities of cytochrome P-450 (CYP) 3A and 2C. Furthermore, we compared the hepatocyte differentiation efficacy of our protocol with another published method, and the results demonstrated that our differentiation strategy could significantly improve the generation of morphological and functional hepatocyte-like cells from porcine iPSCs. In conclusion, this study establishes an efficient method for in vitro generation of functional hepatocytes from porcine iPSCs, which could represent a promising cell source for preclinical testing of cell-based therapeutics for liver failure and for pharmacological applications.
Highlights
Liver failure is the final stage of viral hepatitis, hepatic cirrhosis or cancer, causing a high mortality rate in patients
By using Periodic Acid-Schiff (PAS) staining, glycogen was colored magenta in cytoplasm and we found that approximately 80% of pig iPSCs (piPSCs)-derived hepatocyte-like cells from Method I were positive for glycogen storage, whereas only 40% of Method II cells and almost none of undifferentiated piPSCs positively responded to PAS staining (Fig. 5B)
A variety of hepatic differentiation methods have been established in mouse and human induced pluripotent stem cells (iPSCs) [14,15,16], less attention has been paid to iPSCderived from large animals such as pigs
Summary
Liver failure is the final stage of viral hepatitis, hepatic cirrhosis or cancer, causing a high mortality rate in patients. Liver transplantation has been a successful treatment for end-stage liver disease. Hepatocyte transplantation has been proposed to partially recover liver function, and to extend the lifespan of patients until an organ becomes available [1,2]. The availability of an unlimited number of functional hepatocytes could greatly benefit patients with end-stage liver disease. Embryonic stem (ES) cell-derived hepatocytes have been proposed to be a potential cell source for liver regenerative therapy [3,4]. Induced pluripotent stem cells (iPSCs) have been successfully reprogrammed from somatic cells with defined transcriptional factors [5,6]. IPSCsderived hepatocytes could be utilized as a novel and personalized cell source for future liver disease therapy. Cell replacement therapy for human liver failure has to be preclinically tested in vivo
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