Mahieu-Caputo Dominique, Allain Jean-Etienne, Branger Julie, Coulomb Aurore, Delgado Jean-Paul, Andreoletti Marion, Mainot Sylvie, Frydman René, Leboulch Philippe, Di Santo James P, Capron Frédérique, Weber Anne
Equipe Mixte INSERM 00-20, Laboratoire de Transfert de Gènes dans le Foie: Applications Thérapeutiques, Hôpital Bicêtre, 94270 Le Kremlin Bicêtre and University Paris XI, France.
Hum Gene Ther. 2004 Dec;15(12):1219-28. doi: 10.1089/hum.2004.15.1219.
Transplantation of hepatocytes is a promising alternative to liver transplantation for the treatment of severe liver diseases. However, this approach is hampered by the shortage of donor organs and intrinsic limitations of adult hepatocytes. To investigate whether most of the hurdles faced with adult hepatocytes could be surmounted by the use of human fetal hepatoblasts, we have developed a method to isolate, transduce, and cryopreserve hepatoblasts from human livers at an early stage of development (11-13 weeks of gestation). Cells were characterized in vitro for expression of specific markers, and in vivo for their proliferation and differentiation potential after transplantation into athymic mice. Most of the cells (80-90%) harbored a bipotent phenotype, expressing cytokeratins 8/18, albumin, and CK19. They proliferated spontaneously in culture and were efficiently transduced by a beta-galactosidase-expressing retrovirus (90%). After transplantation, cryopreserved cells engrafted into the liver of athymic mice and proliferated, resulting in up to 10% repopulation. Engrafted cells expressed markers of differentiated adult hepatocytes including albumin, alpha1-antitrypsin, cytochrome P450 3A4, and alpha-glutathione-S-transferase. When retrovirally transduced before transplantation they expressed the transgene in vivo. In summary, early human fetal hepatoblasts engraft, proliferate, and mature in athymic mouse liver, without conditioning the donor.
肝细胞移植是治疗严重肝脏疾病的一种有前景的肝移植替代方法。然而，这种方法受到供体器官短缺和成年肝细胞固有局限性的阻碍。为了研究使用人类胎儿肝母细胞是否可以克服成年肝细胞面临的大多数障碍，我们开发了一种方法，从发育早期（妊娠11 - 13周）的人类肝脏中分离、转导和冷冻保存肝母细胞。对细胞进行体外特定标志物表达的表征，并在体内研究其移植到无胸腺小鼠后增殖和分化的潜力。大多数细胞（80 - 90%）具有双能表型，表达细胞角蛋白8/18、白蛋白和CK19。它们在培养中自发增殖，并被表达β-半乳糖苷酶的逆转录病毒有效转导（90%）。移植后，冷冻保存的细胞植入无胸腺小鼠的肝脏并增殖，导致高达10%的肝组织再填充。植入的细胞表达分化的成年肝细胞标志物，包括白蛋白、α1 -抗胰蛋白酶、细胞色素P450 3A4和α-谷胱甘肽-S-转移酶。在移植前进行逆转录病毒转导时，它们在体内表达转基因。总之，早期人类胎儿肝母细胞在无胸腺小鼠肝脏中植入、增殖并成熟，无需对供体进行预处理。
