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Introduction: Differentiation of hepatocytes and culture methods have been investigated in dogs as a tool to establish liver transplant and drug metabolism examination systems. However, mass culture techniques for canine hepatocytes (cHep) have not been investigated, and it is necessary to construct a suitable culture system. Recently, a protocol called Bud production has attracted attention, and a mixed culture of human and mouse hepatocytes, stem cells, and artificial blood vessels significantly improved the size and formation ratio of spheroids. The purpose of this study was to investigate and improve the in vitro culture of cHep by mixing canine adipose-derived mesenchymal stem cells (cASCs) and human umbilical vein endothelial cells (HUVECs). Methods: Spheroid formation ratio and histological examination were evaluated among four culture methods, including cHep alone, two-mix (cHep + cASCs and cHep + HUVEC), and three-mix (cHep + HUVEC + cASCs), on days 0, 4, and 7. Expression levels of liver-related genes (ALB, AFP, α1-AT, CDH1, CYP2E1, CYP3A12, and TAT) were evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Protein expression of albumin, vimentin, and von Willebrand Factor (vWF) was investigated to confirm the location of the hepatocytes. Results: The ratio of spheroid formation was 60.2% in the three-mix culture and was significantly improved compared with cHep alone (5.9%) and two-mix; cHep + cASCs (36.2%) and cHep + HUVEC (26.4%) (P

Introduction: Hepatocytes, which account for the majority of liver tissue, are derived from the endoderm and become hepatocytes via differentiation of hepatic progenitor cells. Induced hepatocyte-like (iHep) cells and artificial liver tissues are expected to become useful, efficient therapies for severe and refractory liver diseases and to contribute to drug discovery research. The establishment of iHep cell lines are needed to carry out liver transplants and assess liver toxicity in the rising number of dogs affected by liver disease. Recently, direct conversion of non-hepatocyte cells into iHep cells was achieved by transfecting mouse adult fibroblasts with the Forkhead box protein A1 (Foxa1) and hepatocyte nuclear factor 4 homeobox alpha (Hnf4α) genes. Here, we applied this conversion process for the differentiation of canine bone marrow stem cells (cBMSCs) into hepatocyte-like cells. Methods: Bone marrow specimens were collected from four healthy Beagle dogs and used to culture cBMSCs in Dulbecco's Modified Eagle's Medium (DMEM). The cBMSCs displayed the following characteristic features: plastic adherence; differentiation into adipocytes, osteoblasts and chondrocytes; and a cell surface antigen profile of CD29 (+), CD44 (+), CD90 (+), CD45 (−), CD34 (−) and CD14 (−), or CD11b (−) and CD79a (−), or CD19 (−) and HLA class II(−). The cBMSCs were seeded in a collagen I-coated plate and cultured in DMEM with 10% fetal bovine serum and transfected with retroviruses expressing Foxa1 and Hnf4α the following day. Canine iHep cells were differentiated from cBMSCs in culture on day 10, and were analyzed for morphology, RNA expression, immunocytochemistry, urea production, and low-density lipoprotein (LDL) metabolism. Results: The cBMSCs expressed CD29 (98.06 ± 1.14%), CD44 (99.59 ± 0.27%) and CD90 (92.78 ± 4.89%), but did not express CD14 (0.47 ± 0.29%), CD19 (0.44 ± 0.39%), CD34 (0.33 ± 0.25%), CD45 (0.46 ± 0.34%) or MHC class II (0.54 ± 0.40%). The iHep cells exhibited morphology that included circular to equilateral circular shapes, and the formation of colonies that adhered to each other 10 days after Foxa1 and Hnf4α transfection. Quantitative RT-PCR analysis showed that the expression levels of the genes encoding albumin (ALB) and cadherin (CDH) in iHep cells on day 10 were increased approximately 100- and 10,000-fold, respectively, compared with cBMSCs. Corresponding protein expression of ALB and epithelial-CDH was confirmed by immunocytochemistry. Important hepatic functions, including LDL metabolic ability and urea production, were increased in iHep cells on day 10. Conclusion: We successfully induced cBMSCs to differentiate into functional iHep cells. To our knowledge, this is the first report of canine liver tissue differentiation using Foxa1 and Hnf4α gene transfection. Canine iHep cells are expected to provide insights for the construction of liver models for drug discovery research and may serve as potential therapeutics for canine liver disease.