Establishment of differentiation protocols for mouse embryonic stem cells into hepatocytes and insulin producing islet β cells.

(1)小鼠ES细胞转录因子表达调控体系的建立我们从拟胚体中亚克隆了Mix和Hex等中内胚层细胞特异性转录因子的表达调控质粒。这些转录因子在Tet-ON系统的调控下表达。由于将绿色荧光蛋白（GFP）编码序列连接在内部核糖体进入位点（IRES）序列之后，因此可将转录因子优先表达的靶细胞确定为表达绿色荧光蛋白（GFP）的细胞。转染该质粒后产生转基因小鼠ES细胞。(2)小鼠ES细胞体外分化为成熟肝细胞和产生胰岛素的胰岛β细胞在分离来自如前所述产生的转基因小鼠ES细胞的GFP阳性细胞（其通过在培养基中施用多西环素表达Mix）后， ...更多信息 在通过几种生长因子（HGF、FGF 2、ATRA）和几种细胞外基质（I型、IV型胶原、层粘连蛋白、基质胶）的组合鉴定的几种条件下培养。采用RT-PCR方法检测分化培养过程中胚层和内胚层标记基因的表达。虽然在分离的细胞中鉴定了几种中胚层标志物如Goalbumid和Brachury以及几种内胚层标志物如GATA 4和FoxA 2的表达，但未鉴定出肝细胞标志物如甲胎蛋白、白蛋白的表达。此外，未测定Glut-2和Pdx-1的表达，而在分离的Mix表达细胞中检测到胰岛素的表达。因此，在我们的实验中未证实肝细胞和产生胰岛素的胰岛β细胞的充分分化结果。另一方面，我们鉴定了来自小鼠ES细胞的细胞中肝细胞标志物的表达，所述细胞来自与来自小鼠胎肝的Thy-1阳性间充质细胞共培养的小鼠ES细胞。我们还确定了这些分化的细胞，使用相差显微镜和电子显微镜的肝细胞的形态特征。(3)小鼠胚胎干细胞分化的肝细胞样细胞的移植我们将LacZ基因标记的小鼠胚胎干细胞分化的肝细胞样细胞移植到白喉毒素致肝损伤的转基因小鼠体内。我们不仅证实了移植的肝细胞样细胞进入受体肝脏，而且还证实了受体小鼠致死性肝损伤的死亡率的改善。少

(1) Establishment of regulatory system for the expression of transcription factors in mouse ES cellsWe produced regulatory plasmid designed to express transcription factors specific for mesoendodermal cells such as Mix and Hex, which were subcloned from embryoid bodies. These transcription factors were expressed under the control of Tet-ON system. The target cells in which transcription factors were preferentially expressed could be determined as green fluorescent protein (GFP) expressing cells because GFP coding sequence was ligated after the sequence of internal ribosomal entry site (IRES). Transgenic mouse ES cells were produced after the transfection of this plasmid.(2) In vitro differentiation of mouse ES cells into mature hepatocytes and insulin producing islet β cellsAfter the isolation of GFP positive cells derived from transgenic mouse ES cells produced as previously described, which express Mix by the administration of Doxcycline in the culture medium, GFP positive cells were … More cultured under several conditions identified by the combination of several growth factors (HGF, FGF2, ATRA) and also several extra-cellular matrices (Collagen type I, IV, Laminin, Matrigel). RT-PCR was performed to identify the expression of mesodermal and endodermal marker genes in the cells during the differentiation culture. Although the expression of several mesodermal markers such as Gooscoid and brachury and also several endodermal marker such as GATA4 and FoxA2 were identified in the isolated cells, the expression of hepatocyte marker such as alfa-fetoprotein, albumin were not identified. In addition, the expression of Glut-2 and Pdx-1 were not determined whereas the expression of insulin was detected in the isolated Mix expressing cells. Therefore, the sufficient differentiation findings for hepatocytes and also insulin producing islet β cells were not comfirmed in our experiments. On the other hand, we identified the expression of hepatocyte markers in the cells derived from mouse ES cells co-cultured with Thy-1 positive mesenchymal cells originated from mouse fetal liver. We also identified morphological features as hepatocytes in these differentiated cells using phase contrast microscopy and also electron microscopy.(3) The transplantation of hepatocyte like cells derived from mouse ES cellsWe performed transplantation experiments of hepatocyte-like cells differentiated from mouse ES cells marked with LacZ gene into transgenic mice in which lethal liver damage occurred by the administration of diphtheria toxin. We identified not only the incorporation of transplanted hepatocyte like cells into recipient liver but also the improvement of mortality rate ofrecipient mice suffered from lethal liver damage. Less