<p>Hepatic Differentiation of Marmoset Embryonic Stem Cells and Functional Characterization of ESC-Derived Hepatocyte-Like Cells</p>

Abstract

Background: Primary human hepatocytes (PHHs) are the ideal candidates for studying critical liver functions such as drug metabolism and toxicity. However, as they are isolated from discarded livers that are unsuitable for transplantation, they possess limited expansion ability in vitro and their enzymatic functions deteriorate rapidly because they are often of poor quality. Therefore, there is a compelling reason to find reliable alternative sources of hepatocytes. Methods: In this study, we report on efficient and robust differentiation of embryonic stem cells (ESC) from the common marmoset Callithrix jacchus into functional hepatocyte-like cells (HLC) using a simple, and reproducible three-step procedure. ESC-derived HLCs were examined by morphological analysis and tested for their expression of hepatocyte-specific markers using a combination of immunohistochemistry, RT-PCR, and biochemical assays. Primary human hepatocytes were used as controls. Results: ESC-derived HLCs expressed each of the hepatocyte-specific markers tested, including albumin; α-fetoprotein; asialoglycoprotein receptor 1; α-1 antitrypsin; hepatocyte nuclear factors 1α and 4; cytokeratin 18; hepatocyte growth factor receptor; transferrin; tyrosine aminotransferase; alkaline phosphatase; c-reactive protein; cytochrome P450 enzymes CYP1A2, CYP2E1 and CYP3A4; and coagulation factors FVII and FIX. They were functionally competent as demonstrated by biochemical assays in addition to producing urea. Conclusion: Our data strongly suggest that marmoset HLCs possess characteristics similar to those of PHHs. They could, therefore, be invaluable for studies on drug metabolism and cell transplantation therapy for a variety of liver disorders. Because of the similarities in the anatomical and physiological features of the common marmoset to that of humans, Callithrix jacchus is an appropriate animal model to study human disease conditions and cellular functions.

Common Marmoset Cell Lines and Their Applications in Biomedical Research

“…Recently, Aravalli et al have carried out efficient hepatic differentiation of marmoset embryonic stem cells (ESCs) into functional hepatocyte-like cells (HLC) and demonstrated that the generated HLCs possessed specific characteristics similar to those of primary human hepatocytes. HLCs might be an optimal model for research in cell therapy of human liver diseases including hepatotropic virus infections, while the expression of CYP genes involved in the breakdown of various toxic molecules and chemicals also makes HLCs suitable for research of drug metabolism [ 95 ]. The potential for neural differentiation has been reported by Shimada et al [ 96 ].…”

Utility of Common Marmoset (Callithrix jacchus) Embryonic Stem Cells in Liver Disease Modeling, Tissue Engineering and Drug Metabolism

“…Moreover, they can be converted from the primed to a naive-like state using transgenes to increase their pluripotency in vitro [ 47 ]. cjESCs were recently differentiated into highly functional hepatocyte-like cells (cjESC-HLCs) [ 48 ], which would be valuable for in vitro studies on infectious diseases, regenerative medicine and drug metabolism. While it has been shown that iPSCs can allograft into the putamen of cynomolgus monkeys without immunosuppression [ 49 ], cjESC-derived cells were only tested using immunosuppressive agents such as tacrolimus [ 50 ].…”

“…To overcome these bottlenecks, successful attempts have been made to model HCV infection in HLCs isolated from human-induced pluripotent stem cells (iPSCs) [ 84 , 85 ] and human ESCs [ 85 ]. Since C. jacchus is a good surrogate for HCV, the common marmoset could be an appropriate animal model to investigate hepatitis viruses in in vitro studies; and recently generated cjESC-HLCs [ 48 ] could be very valuable tools for in vitro experiments.…”

“…cjESCs have a distinct advantage over their human counterparts as they are not restricted for scientific studies. cjESCs can be cultured for more than 50 population doublings, and cjESC-HLCs can be maintained in culture for a prolonged period of time without losing their phenotypic characteristics, including the expression of the asialoglycoprotein receptor, albumin, tyrosine aminotransferase, cytokeratins, CYP enzymes, and coagulation factors VII and IX [ 48 ]. High-quality cjESC-HLCs are also good substitutes for in vitro studies when compared to PHHs because they have low variability, if any, and can be easily generated in large numbers using the unlimited supply of cjESCs.…”

“…cjESC-HLCs were recently shown to express three CYP enzymes (CYP1A2, CYP2E1 and CYP3A4) at levels comparable to those of their human counterparts [48]. Among these, CYP1A2, which accounts for about 13% of all CYP450 enzymes in the liver, is a major enzyme that metabolizes both endogenous compounds such as melatonin, estradiol, bilirubin and arachidonic acid, as well as several clinical drugs, including analgesics and antipyretics [198].…”