Efficient In Vitro Induction of Hepatocyte-like Cells From Embryonic Stem Cells

我们以前证明，小鼠ES细胞，基因标记的绿色荧光蛋白，有能力在体外分化成肝细胞样细胞。在2004年，我们还研究了小鼠ES细胞是否可以在体内分化为肝细胞样细胞。小鼠腹腔注射四氯化碳（CCl_4，0.5ml/Kg，每周2次）5周。第二次注射CCl_4后，将10^5个未分化的ES细胞或10个胚状体（EBs）移植到小鼠脾脏中。在移植前，使用500个未分化的ES细胞在20 μl不含LIF的培养基中的5天悬滴培养来制备EB。在移植后第10、20和30天进行组织学评价。在PD 10时，在肝小叶的门静脉周围区域发现GFP免疫阳性细胞，之后在PD 20时发现这些细胞扩散到小叶中。然而，他们在PD 30时消失了。在CCl_4未处理的小鼠中，在PD 10、20或30的肝脏中未发现GFP免疫阳性细胞。双重免疫荧光研究显示GFP免疫阳性细胞也对白蛋白、细胞角蛋白18和HNF 4 α呈阳性。此外，GFP阳性细胞的小叶内分布在ES-和EB-移植小鼠之间相似，并且在两种小鼠中均观察到分化为表达白蛋白、细胞角蛋白18和HNF 4 α的肝细胞样细胞的ES细胞。我们的研究结果表明，基于ES的细胞疗法对肝脏疾病可能有用。

We previously demonstrated that mouse ES cells, genetically labeled with GFP, have an ability to differentiate into hepatocyte-like cells in vitro. In 2004, we also investigated whether mouse ES cells could differentiate into hepatocyte-like cells in vivo. Mice were treated with carbon tetrachloride (CCl_4, intraperitoneal injection of 0.5 ml/Kg, twice a week) for 5 weeks. Following the second administration of CCl_4, 10^5 undifferentiated ES cells or 10 embryoid bodies(EBs) were transplanted into the mice spleens. Prior to transplantion, the EBs were prepared using a 5-day hanging-drop culture of 500 undifferentiated ES cells in 20 μl of culture medium without LIF. Histological evaluations were performed on post-transplantation day(PD) 10, 20, and 30. GFP-immunopositive cells were found in the periportal regions of the liver lobules on PD 10, after which those cells were found spread into the lobules on PD 20. However, they disappeared by PD 30. In CCl_4-untreated mice, GFP-immunopositive cells were not found in the liver on PD 10, 20, or 30. A double immunofluorescent study revealed that GFP-immunopositive cells were also positive for albumin, cytokeratin 18, and HNF4α. Further, the intralobular distribution of GFP-positive cells was similar between ES- and EB-transplanted mice, and ES cells that had differentiated into hepatocyte-like cells expressing albumin, cytokeratin 18, and HNF4α were observed in both. Our results suggest the possible usefulness of ES-based cell therapy for liver diseases.