Allain Jean-Etienne, Dagher Ibrahim, Mahieu-Caputo Dominique, Loux Nathalie, Andreoletti Marion, Westerman Karen, Briand Pascale, Franco Dominique, Leboulch Philippe, Weber Anne
Equipe Mixte Inserm 00-20, Laboratoire de Transfert de Gènes Dans le Foie: Applications Thérapeutiques, Hôpital Antoine-Béclère, 157 Rue de la Porte de Trivaux, 92141 Clamart, France.
Proc Natl Acad Sci U S A. 2002 Mar 19;99(6):3639-44. doi: 10.1073/pnas.062038599.
Liver regeneration after partial hepatectomy results primarily from the simple division of mature hepatocytes. However, during embryonic and fetal development or in circumstances under which postnatal hepatocytes are injured, organ regeneration is believed to occur from a compartment of epithelial liver stem or progenitor cells with biliary and hepatocytic bipotentiality. The ability to identify, isolate, and transplant epithelial liver stem cells from fetal liver would greatly facilitate the treatment of hepatic diseases currently requiring orthotopic liver transplantation. Here we report the identification and immortalization by retrovirus-mediated transfer of the simian virus 40 large T antigen gene of primate fetal epithelial liver cells with a dual hepatocytic biliary phenotype. These cells grow indefinitely in vitro and express the liver epithelial cell markers cytokeratins 8/18, the hepatocyte-specific markers albumin and alpha-fetoprotein, and the biliary-specific markers cytokeratins 7 and 19. Bipotentiality of gene expression was confirmed by clonal analysis initiated from single cells. Endogenous telomerase also is expressed constitutively. After orthotopic transplantation via the portal vein, approximately 50% of the injected cells integrated into the liver parenchyma of athymic mice without tumorigenicity. Three weeks after transplantation, cells having seeded in the liver parenchyma expressed both albumin and alpha-fetoprotein but had lost expression of cytokeratin 19. These results provide strong evidence for the existence of a bipotent epithelial liver stem cell in nonhuman primates. This unlimited source of donor cells also should enable the establishment of a model of allogenic liver cell transplantation in a large animal closely related to humans and shed light on important questions related to liver organogenesis and differentiation.
部分肝切除术后的肝脏再生主要源于成熟肝细胞的简单分裂。然而，在胚胎和胎儿发育期间或出生后肝细胞受损的情况下，器官再生被认为是由具有胆管和肝细胞双潜能的上皮性肝干细胞或祖细胞群发生的。从胎儿肝脏中识别、分离和移植上皮性肝干细胞的能力将极大地促进目前需要原位肝移植的肝脏疾病的治疗。在此，我们报告通过逆转录病毒介导的猿猴病毒40大T抗原基因转移，对具有双肝细胞胆管表型的灵长类胎儿上皮性肝细胞进行识别和永生化。这些细胞在体外无限增殖，并表达肝上皮细胞标志物细胞角蛋白8/18、肝细胞特异性标志物白蛋白和甲胎蛋白，以及胆管特异性标志物细胞角蛋白7和19。通过单细胞克隆分析证实了基因表达的双潜能性。内源性端粒酶也持续表达。通过门静脉进行原位移植后，大约50%的注入细胞整合到无胸腺小鼠的肝实质中，且无致瘤性。移植后三周，植入肝实质的细胞同时表达白蛋白和甲胎蛋白，但失去了细胞角蛋白19的表达。这些结果为非人类灵长类动物中存在双潜能上皮性肝干细胞提供了有力证据。这种无限的供体细胞来源也应能建立与人类密切相关的大型动物同种异体肝细胞移植模型，并阐明与肝脏器官发生和分化相关的重要问题。
