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.

中文翻译：

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Foxa1 和 Hnf4a 将间充质干细胞转化为犬肝细胞样细胞。

介绍

占肝组织大部分的肝细胞来源于内胚层并通过肝祖细胞分化成为肝细胞。诱导肝细胞样 (iHep) 细胞和人工肝组织有望成为治疗严重和难治性肝病的有用、有效的疗法，并有助于药物发现研究。需要建立 iHep 细胞系来进行肝移植并评估越来越多的受肝病影响的狗的肝毒性。最近，通过用 Forkhead box 蛋白 A1 ( Foxa1 ) 和肝细胞核因子 4 同源框 α ( Hnf4α )转染小鼠成体成纤维细胞，可以将非肝细胞直接转化为 iHep 细胞。) 基因。在这里，我们将这种转化过程应用于将犬骨髓干细胞 (cBMSCs) 分化为肝细胞样细胞。

方法

从四只健康的 Beagle 犬收集骨髓标本，并用于在 Dulbecco 改良 Eagle 培养基 (DMEM) 中培养 cBMSC。cBMSCs表现出以下特征：塑料粘附；分化成脂肪细胞、成骨细胞和软骨细胞；CD29 (+)、CD44 (+)、CD90 (+)、CD45 (-)、CD34 (-) 和 CD14 (-) 或 CD11b (-) 和 CD79a (-) 或 CD19 的细胞表面抗原谱(-) 和 HLA II 类 (-)。将 cBMSCs 接种在胶原蛋白 I 包被的板中，在含有 10% 胎牛血清的 DMEM 中培养，并用表达Foxa1和Hnf4α的逆转录病毒转染次日。在第 10 天，将犬 iHep 细胞从培养的 cBMSCs 中分化出来，并分析其形态、RNA 表达、免疫细胞化学、尿素产生和低密度脂蛋白 (LDL) 代谢。

结果

cBMSCs 表达 CD29 (98.06 ± 1.14%)、CD44 (99.59 ± 0.27%) 和 CD90 (92.78 ± 4.89%)，但不表达 CD14 (0.47 ± 0.29%)、CD19 (0.44 ± 0.39%)、CD34 (0.33 ± 0.25%)、CD45 (0.46 ± 0.34%) 或 MHC II 类 (0.54 ± 0.40%)。iHep 细胞的形态包括圆形到等边圆形，并在Foxa1和Hnf4α转染 10 天后形成相互粘附的集落。定量 RT-PCR 分析显示，编码白蛋白 ( ALB ) 和钙粘蛋白 ( CDH ) 基因的表达水平) 与 cBMSCs 相比，第 10 天 iHep 细胞中的含量分别增加了约 100 倍和 10,000 倍。通过免疫细胞化学证实了 ALB 和上皮-CDH 的相应蛋白表达。在第 10 天，iHep 细胞中的重要肝功能，包括 LDL 代谢能力和尿素产生增加。

结论

我们成功诱导 cBMSCs 分化为功能性 iHep 细胞。据我们所知，这是使用Foxa1和Hnf4α基因转染的犬肝组织分化的第一份报告。犬 iHep 细胞有望为构建用于药物发现研究的肝脏模型提供见解，并可能作为犬肝病的潜在治疗剂。