Treatment of liver failure by transplantation of hepatocytes derived from mouse and monkey ES cells and human umbilical cord blood

1)猴胚胎干细胞具有与人类胚胎干细胞相似的特性，可作为临床前研究的替代模型。在培养猴ES细胞形成的类胚体时，观察到许多肝细胞相关基因的表达，包括细胞色素P450(Cyp)3a和Cyp7a1。用白蛋白(ALB)、细胞角蛋白-8/18和A1-抗胰蛋白酶抗体对发育中的EBS中的肝细胞进行免疫细胞化学观察。猴ES细胞在体外向肝系分化的潜力促使我们研究猴子EB细胞的可移植性。作为评估再生潜力的初步方法，我们将EB细胞移植到免疫缺陷的尿激酶型纤溶酶原激活物转基因小鼠中，这些小鼠经历了肝功能衰竭。移植后，在小鼠肝脏内可观察到表达猴ALB的肝细胞集落。荧光原位杂交显示再生的肝细胞为…更多来自于移植的猴EB细胞与受体小鼠肝细胞的细胞融合。相比之下，在未分化的ES细胞移植后，受体肝脏中既没有观察到细胞融合，也没有观察到肝细胞的再繁殖。这些结果表明，在发育中的猴EBS中，分化的细胞不污染ES细胞，通过与受体小鼠肝细胞的细胞融合产生功能肝细胞，并重新植入受损的小鼠肝脏。2)我们以前报道过肝细胞可以通过类胚体(EB)形成的方式从胚胎干细胞分化而来，并可以移植到小鼠肝脏中。然而，EB来源的细胞移植经常导致受体肝脏形成畸胎瘤。在这项研究中，我们清除了EB突起中的致瘤细胞，并检测了浓缩的ES细胞来源的肝细胞移植到受损肝脏的效果。培养第15天，EBS部分解体并传代培养。通过肝细胞标志物的表达检测传代细胞中的肝细胞。Percoll不连续梯度离心法去除EB细胞中污染的未分化细胞。此外，使用血小板/内皮细胞黏附分子(PECAM)-1和Mac-1抗体通过磁性细胞分选消除未分化细胞、内皮细胞和巨噬细胞。然后将这些丰富的ES细胞来源的肝细胞移植到受损的小鼠肝脏中。Percoll离心法和PECAM-1抗体将表达Oct-3/4的未分化细胞从EB来源的细胞中剔除。ES细胞来源的肝细胞在传代培养过程中表现出肝脏相关基因的表达、尿素和糖原的合成以及结构特征。移植研究表明，丰富的ES细胞来源的肝细胞整合到受损的小鼠肝脏中，没有产生畸胎瘤。当ES细胞来源的肝细胞被移植到CCl4损伤的肝脏中时，肝功能随后得到改善。小鼠ES细胞可通过EB形成分化为有功能的肝细胞。从EBS中去除未分化的细胞为肝功能衰竭提供了可移植的细胞，而没有致瘤性。较少

1) Monkey embryonic stem (ES) cells have characteristics that are similar to human ES cells, and might be useful as a substitute model for preclinical research. When embryoid bodies (EBs) formed from monkey ES cells were cultured, expression of many hepatocyte-related genes including cytochrome P450 (Cyp) 3a and Cyp7a1 was observed. Hepatocytes were immunocytochemically observed using antibodies against albumin (ALB), cytokeratin-8/18, and a1-antitrypsin in the developing EBs. The in vitro differentiation potential of monkey ES cells into the hepatic lineage prompted us to examine the transplantability of monkey EB cells. As an initial approach to assess the repopulation potential, we transplanted EB cells into immunodeficient urokinasetype plasminogen activator transgenic mice that undergo liver failure. After transplantation, the hepatocyte colonies expressing monkey ALB were observed in the mouse liver. Fluorescence in-situ hybridization revealed that the repopulating hepatocytes ar … More ise from cell fusion between transplanted monkey EB cells and recipient mouse hepatocytes. In contrast, neither cell fusion nor repopulation of hepatocytes was observed in the recipient liver after undifferentiated ES cell transplantation. These results indicate that the differentiated cells in developing monkey EBs, but not contaminating ES cells, generate functional hepatocytes by cell fusion with recipient mouse hepatocytes, and repopulate injured mouse liver.2) We previously reported that hepatocytes can be differentiated from embryonic stem (ES) cells by way of embryoid body (EB) formation and are transplantable into the mouse liver. However, the transplantation of EB-derived cells frequently resulted in teratoma formation in the recipient liver. In the present study, we eliminated the tumorigenic cells from EB outgrowths and examined the effects of enriched ES-cell-derived hepatocyte transplantation into an injured liver. On day 15 in culture, the EBs were partially disaggregated and subcultured. Hepatocytes in the subcultured cells were examined by the expression of hepatocyte markers. Undifferentiated cells contaminating in the EB-derived cells were eliminated by Percoll discontinuous gradient centrifugation. Furthermore, undifferentiated cells, endothelial cells, and macrophages were eliminated by magnetic cell sorting using platelet/endothelial cell adhesion molecule (PECAM)-1 and Mac-1 antibodies. These enriched ES-cell-derived hepatocytes were then transplanted into the injured mouse liver. Percoll centrifugation and PECAM-1 antibodies eliminated the undifferentiated cells expressing Oct-3/4 from the EB-derived cells. ES-cell-derived hepatocytes showed expression of liver-related genes, synthesis of urea and glycogen, and structural characteristics during subculture. A transplantation study showed that the enriched ES-cell-derived hepatocytes integrated into the injured mouse liver and produced no teratomas. When the ES-cell-derived hepatocytes were transplanted into a CCl4-injured liver, the liver function was subsequently improved. Functional hepatocytes can be differentiated from mouse ES cells by way of EB formation. The elimination of undifferentiated cells from the EBs provides transplantable cells for liver failure without tumorigenicity. Less