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

小鼠和猴 ES 细胞以及人脐带血来源的肝细胞移植治疗肝衰竭

• 批准号: 16390357
• 负责人: TERAMOTO Kenichi
• 金额: $ 8.9万
• 依托单位: Tokyo medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390357/
• 关键词: Cynomolgus; Embryonic stem cells; Hepatocyte differentiation; Transgenic mouse; umbilical cord blood; liver failure; hepatocyte transplantation; teratoma; マウスES細胞; 胚様体; 肝細胞分化; 細胞移植; 肝細胞

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

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。当培养由猴ES细胞形成的胚状体（EBs）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Cyp 7a 1。肝细胞免疫细胞化学观察使用抗体对白蛋白（ALB），细胞角蛋白-8/18，和α 1-抗胰蛋白酶在发展中的EB。猴胚胎干细胞在体外向肝细胞系分化的潜能促使我们研究猴EB细胞的可移植性。作为评估再增殖潜力的初步方法，我们将EB细胞移植到免疫缺陷型尿激酶型纤溶酶原激活剂转基因小鼠中，这些小鼠经历了肝功能衰竭。移植后，在小鼠肝脏中观察到表达猴ALB的肝细胞集落。荧光原位杂交显示，再生的肝细胞 ...更多信息 伊势于移植的猴EB细胞和受体小鼠肝细胞之间的细胞融合。相反，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增殖。这些结果表明，在发育中的猴EB，但不污染ES细胞的分化细胞，通过与受体小鼠肝细胞融合产生功能性肝细胞，并重新填充损伤的小鼠肝脏。2）我们以前报道过，肝细胞可以通过胚状体（EB）形成的方式从胚胎干（ES）细胞分化，并可移植到小鼠肝脏中。然而，EB衍生细胞的移植经常导致受体肝脏中畸胎瘤的形成。在本研究中，我们消除了EB副产物的致瘤细胞，并研究了富集ES细胞来源的肝细胞移植到受损肝脏的效果。在培养的第15天，EB部分分解并传代培养。通过肝细胞标志物的表达检测传代细胞中的肝细胞。通过Percoll不连续梯度离心去除EB衍生细胞中污染的未分化细胞。此外，通过使用血小板/内皮细胞粘附分子（PECAM）-1和Mac-1抗体的磁性细胞分选，消除未分化细胞、内皮细胞和巨噬细胞。然后将这些富集的ES细胞衍生的肝细胞移植到损伤的小鼠肝脏中。Percoll离心和PECAM-1抗体从EB衍生的细胞中消除了表达Oct-3/4的未分化细胞。ES细胞衍生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，富集的ES细胞衍生的肝细胞整合到损伤的小鼠肝脏中，并且不产生畸胎瘤。当ES细胞来源的肝细胞移植到CCl 4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形成分化为功能性肝细胞。从EB中消除未分化的细胞为肝衰竭提供了无致瘤性的可移植细胞。少

项目成果

Enrichment of hepatocytes differentiated from mouse embryonic stem cells as a transplantable source

• DOI: 10.1097/01.tp.0000153637.44069.c6
• 发表时间: 2005-03-15
• 期刊: TRANSPLANTATION
• 影响因子: 6.2
• 作者: Kumashiro, Y;Asahina, K;Teraoka, H
• 通讯作者: Teraoka, H

Expression of the liver-specific gene Cyp7al reveals hepatic differentiation in embryoid bodies derived from mouse embryonic stem cells

肝脏特异性基因 Cyp7al 的表达揭示了小鼠胚胎干细胞胚状体的肝分化

• 发表时间: 2004
• 期刊: Genes to Cells 9
• 作者: Kinji Asahina;Hiroaki Fujimori;Keiko Shimizu-saito;Yuji Kumashiro;Kentaro Okamura;Yujiro Tanaka;Kenichi Teramoto;Shigeki Arii;Hir
• 通讯作者: Hir

ES細胞からの肝細胞分化の試み

尝试区分肝细胞和 ES 细胞

• 发表时间: 2004
• 期刊: 低温医学 30(2)
• 作者: 寺本研一;神代祐至;入江工;有井滋樹
• 通讯作者: 有井滋樹

Teratoma formation and hepatocyte differentiation in mouse liver transplanted with mouse embryonic stem cell-derived embryoid bodies.

• DOI: 10.1016/j.transproceed.2004.12.120
• 发表时间: 2005
• 期刊: Transplantation proceedings
• 影响因子: 0.9
• 作者: K. Teramoto;Y. Hara;Y. Kumashiro;Ryoko Chinzei;Yujiro Tanaka;K. Shimizu‐saito;K. Asahina;H. Teraoka;S. Arii

Generation of hybrid hepatocytes by cell fusion from monkey embryoid body cells in the injured mouse liver

• DOI: 10.1007/s00418-005-0065-1
• 发表时间: 2006-03-01
• 期刊: HISTOCHEMISTRY AND CELL BIOLOGY
• 影响因子: 2.3
• 作者: Okamura, K;Asahina, K;Teraoka, H
• 通讯作者: Teraoka, H